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Taxonomically, the pathogenic Gram-positive rods encompass pathogens from both the Actinobacteria and Firmicutes. Typically, however, these pathogens are categorized phenotypically into endospore formers (Bacillus and Clostridium) and non-spore forming rods. The non-spore formers are further divided into acid fast (Mycobacterium) and not acid fast (exemplified here by Listeria). The selected pathogens described here are just a few of the most relevant to this course.
15.1.1: Gram Positive Rods - Biology
Infectious lymphocele is a rare post-operative complication of abdominal surgery, and few studies have focused on its causative organisms. The aim in this research is to clarify microbiology and appropriate empiric treatment of infective lymphocele.
We performed a single center, retrospective observational study between April 2000 and March 2018 with a case review and literature search. Data were collected in a chart review.
Twenty-four cases were founded in our institution. 153 cases, including 16 cases from our institution, that detected causative organisms was also analyzed. Infectious lymphocele was found to occur post gynecological/urological surgery in cancer patients. We also reported that bacteremia incidence and the mortality rate of infectious lymphocele cases were very low. The major sites of infectious lymphocele were pelvis or inguinal area.
Our case series and literature review showed Gram positive cocci were the major causative organisms, with Staphylococcus aureus constituting one third of them (53/153 cases). Streptococcus species (26/153cases) and coagulase negative Staphylococci (17/153 cases) were the second and third most detected organisms.
In gynecologic and urologic cancer patients, Gram positive cocci were the most common organisms causing lymphocele infection. Gram-positive coverage might be reasonable for empiric therapy in infectious lymphocele.
15.1.1: Gram Positive Rods - Biology
Parapneumonic pleural effusions/empyema (PPE/PE) are severe complications of community-acquired pneumonia. We investigated the bacterial aetiology and incidence of paediatric PPE/PE in Germany after the introduction of universal pneumococcal conjugate vaccine (PCV) immunization for infants.
Children <18 years of age hospitalized with pneumonia-associated PPE/PE necessitating pleural drainage or persisting >7 days were reported to the German Surveillance Unit for Rare Diseases in Childhood between October 2010 and June 2017. All bacteria detected in blood or pleural fluid (by culture/PCR) were included, with serotyping for Streptococcus pneumoniae.
The median age of all 1447 PPE/PE patients was 5 years (interquartile range 3–10). In 488 of the 1447 children with PPE/PE (34%), 541 bacteria (>40 species) were detected. Aerobic gram-positive cocci accounted for 469 of 541 bacteria detected (87%) these were most frequently Streptococcus pneumoniae (41%), Streptococcus pyogenes (19%) and Staphylococcus aureus (6%). Serotype 3 accounted for 45% of 78 serotyped S. pneumoniae strains. Annual PPE/PE incidence varied between 14 (95%CI 12–16) and 18 (95%CI 16–21) PPE/PE per million children. Incidence of S. pneumoniae PPE/PE decreased from 3.5 (95%CI 2.5–4.6) per million children in 2010/11 to 1.5 (95%CI 0.9–2.4) in 2013/14 (p 0.002), followed by a re-increase to 2.2 (95%CI 1.5–3.2) by 2016/17 (p 0.205).
In the era of widespread PCV immunization, cases of paediatric PPE/PE were still caused mainly by S. pneumoniae and, increasingly, by S. pyogenes. The re-increase in the incidence of PPE/PE overall and in S. pneumoniae-associated PPE/PE indicates ongoing changes in the bacterial aetiology and requires further surveillance.
Microbiological Spectrum of Brain Abscess at a Tertiary Care Hospital in South India: 24-Year Data and Review
Intracranial abscesses are life-threatening infections that pose a diagnostic challenge not only to the neurosurgeon but also to the microbiologists. Detailed studies documenting the spectrum of infecting agents involved in brain abscesses are limited from India. Materials and Methods. This is a retrospective analysis of 352 samples from 1987 to 2010 analyzed at a tertiary care hospital in South India from 1987 to 2010, to document the changing trends with time. Results. The age of the patients ranged from 2 to 80 years, a larger number of males being affected. Otogenic infections were the most common cause while cryptogenic abscesses were 20%. Gram stain and culture positivity were 78% each. Gram-positive and negative facultative aerobes and obligate anaerobes were also on the rise. Unusual organisms, like Burkholderia pseudomallei, Salmonella typhi, Nocardia species, Cladosporium bantiana, Fonsecaea pedrosoi, Entamoeba histolytica, and Acanthamoeba were also isolated and/or detected from the brain abscesses aspirate or resected tissue. Summary. New and emerging pathogens associated with brain abscess, especially in immunosuppressed individuals, have renewed the necessity of an early detection, and it will be of great value in appropriate management of patients with brain abscess.
Intracranial abscesses (usually referred to as brain abscess), though uncommon in developed countries, are serious, life-threatening infections [1–3]. Major advances, such as stereotactic neurosurgical procedures, discovery of newer antibiotics, especially metronidazole against anaerobes and ceftriaxone which effectively crosses the blood brain barrier, and newer imaging techniques for early detection of brain abscesses , have lead to a substantial reduction in the mortality [4, 5]. Despite these advances, brain abscess remains a potentially fatal central nervous system (CNS) disease, especially in developing countries [3–9].
Difficulties in the diagnosis of intracranial abscess are mainly due to protean clinical manifestations and similarities in the imaging and morphologic appearance of some intracranial mass lesions, like cystic gliomas and metastases. The frequent delay in making the diagnosis renders this condition a significant challenge for the neurosurgeon [8–10] in the management of the case.
New and emerging pathogens, especially in immunosuppressed individuals, have renewed concern about the diagnosis and treatment of brain abscess and equally pose a challenge to the clinical microbiologist [1, 3, 7, 11]. Meticulous microbiological investigations, including critical microscopic examination for all possible infectious agents and employing detailed microbial investigative armamentarium for the isolation of the organism(s) from the abscess material and from the probable primary site of origin of the infection elsewhere in the body, will definitely aid in the identification of the etiological agent(s) [2, 9–11]. These findings will enable the neurosurgeon and the infectious disease (ID) specialist to treat the brain abscess more rationally and appropriately [5, 7, 8, 10].
It is well worth stating that a brain abscess is not only a neurosurgical emergency but also a microbiological emergency and a diagnostic challenge to both the disciplines [1, 10, 12].
The objectives of this retrospective study were to analyze the microbiological findings in the purulent aspirates and/or tissue obtained from the brain abscesses and discuss the changing and evolving spectrum of infectious agents observed over the past 24 years with a brief review of other studies.
2. Materials and Methods
This is a hospital-based retrospective microbiological analysis of 352 brain abscess materials (purulent aspirates and/or tissue) that were received between 1987 and 2010, by the Microbiology Laboratory, at the Nizam's Institute of Medical Sciences, a tertiary care and a teaching hospital, in South India. The data was analyzed in two groups—group I, between 1987 and 1993 (published data)  and group II between 1994 and 2010, to document the changing trends in the microbial flora and treatment strategies. (1) The demographic and clinical information of the patients was retrieved from the medical records section. Relevant data recorded included the age and sex of the patients, intracranial location of the abscess(es), the probable primary source of the infectious agents leading to the formation of the abscess(es). (2) The microbiology data was retrieved from the microbiology records maintained as a database in the Microbiology Department. (3) Wherever the resected brain abscess tissue was received, the histopathological features were corroborated to complement the culture results.
3. Microbiological Investigations
Specimen collected from a brain abscess (either through a burr hole or craniotomy) during any time of the day and submitted for microbiological investigations was considered as an emergency specimen and was processed immediately in the microbiology laboratory, on priority.
Specimen received between the years 1987 and 1993 (study group I)  was processed only for bacteria (aerobic and anaerobic), by routine microbiological procedures . (i) Gram's stain was carried out on all specimens for bacteria, while Zeihl Neelsen's (ZN) stain for acid fast bacilli (AFB) was done in only one case of tuberculous brain abscess. (ii) Aerobic cultures were performed on 7% sheep blood agar and McConkey agar and incubated at 37°C for 48 hours, before being declared as sterile. All positive cultures were further processed for identification and antibiotic susceptibility patterns . (iii) Anaerobic culture was performed on 7% sheep blood agar plates and incubated in the Dynamicro Gaspak system. Metronidazole disc (5 μg) was placed to observe for antibiotic susceptibility of anaerobes. Isolates susceptible to metronidazole were considered to be anaerobes. Gram's stain of such isolates was carried out to confirm the morphology and the genus of the isolate. Aerotolerance test was performed to demonstrate that these isolates were obligate anaerobes. (iv) The specimen from the tuberculous abscess showed AFB on ZN stain. The material was inoculated on Lowenstein Jensen's (LJ) medium, and the isolate was identified as Mycobacterium tuberculosis (M.tb) based on its rate of growth and susceptibility to para nitro benzoic acid .
The rest of the 302 brain abscess specimens (study group II) were processed for detection of bacteria (aerobes and anaerobes), mycobacteria, fungal pathogens including Nocardia and Actinomycetes, and parasites, by standard microbiological procedures  and by semiautomated identification systems (Mini API and the Vitek 2, bioMérieux, USA). Various detection procedures used were as per standard guidelines . The anaerobic isolates were identified up to the genus level only.
The average number of brain abscess specimens received for microbiological analysis was 21 per year.
Brain abscesses were diagnosed in all decades of life, with the ages ranging from 2 years to 80 years (mean of
) and a male preponderance (male to female ratio was 2.7 : 1). The youngest patient (2 years) developed a frontal lobe abscess with an underlying septicemia and was referred to our institute for further management. The oldest patient was an 80-year-old lady, with chronic suppurative otitis media (CSOM) and a parietal lobe abscess.
Table 1(a) shows a comparison between the two study groups in terms of the location and source of infection of the brain abscess. Tables 1(b) and 1(c) highlight the topographic distribution of the abscesses and the probable source of infection documented in study group II.
). (c) Location and source of infection of multiple brain abscesses in study group II (
There were 310 (88.1%) nontraumatic and 42 (11.9%) posttraumatic brain abscesses in our study. Though solitary abscesses (313/352 88.9%) were more common, multiple abscesses (39/302 12.9%), predominantly otogenic, were noted only in the study group II. There were 6 cases of subdural empyema and 3 cases of extradural abscesses. The rest of the brain abscesses were intracranial, involving the brain parenchyma. The majority of these intracerebral abscesses were located in the parietal region 102/352 (28.9%), followed by the frontal and the temporal lobes (21%).
5. Microbial Spectrum
Staining and microscopy of the brain abscess material (pus and/or tissue) revealed the pathogens in 41/50 (82%)  and 221/302 (73.1%) of the cases in the 2 groups, respectively. In the rest of the cases, only polymorphonuclear cells were seen, indicating an inflammatory process. Microscopy was positive on all the fungal, mycobacterial, Nocardial, and parasitic abscesses. However, the microscopy was negative in 12 bacterial culture-positive cases in group II.
Microbiological cultures for bacteria, mycobacteria, and fungi (included in the study group II only) revealed the etiological agent(s) in 44/50 (88%)  and 203/302 (67.2%) cases in the two groups. The spectrum of organisms isolated from the brain abscesses in both of the groups is compared in Tables 2(a), 2(b), and 2(c).
Gram-positive facultative aerobes were isolated more frequently than the gram-negative aerobes. Among the isolates, 35/44 (79.5%) and 158/203 (77.83%) were facultative aerobes, 7/44 (15.9%) and 32/203 (15.8%) were obligate anaerobes, and 2/44 (4.5%) and 7/203 (3.4%) were mycobacteria. Nocardia species 3/203 (1.5%) and fungal isolates 5/203 (2.5%) were seen only in group II, probably due to the extensive methods employed. The obligate anaerobes were isolated mainly from the brain abscesses originating from the ear, paranasal, and oral infections. Mixed aerobic and anaerobic abscesses were also frequently encountered. There were 30 microscopy-positive abscesses with very small Gram-positive cocci in pairs and or chains, on Gram's stain, resembling anaerobic cocci. However, no organisms could be recovered by any of the culture methods used from these specimens.
5.1. Bacterial Isolates (Tables 2(a) and 2(b))
5.1.1. Facultative Aerobes (180 Single and 13 Mixed Aerobes, Total 193) (Table 2(a))
Staphylococcus aureus (S. aureus) (68/192, 35.2%) was the most common isolate, often as a single isolate. Compared to the group I, the incidence of S. aureus infections was about 4 times more (Table 2(a)). These isolates were usually from brain abscesses associated with CSOM and trauma cases, in the study group II. There were 6/55 methicillin-resistant S. aureus (MRSA) isolates, from brain abscesses following a road traffic accident (RTA), and they were sensitive to vancomycin. The rest of the isolates were methicillin-sensitive S. aureus (MSSA) and sensitive to a broad range of antibiotics.
Enterococcus species (34/193, 17.6%) were isolated frequently from the cases with CSOM. Compared to the study group I, Enterococcus species appeared to be significant emerging pathogens of brain abscess at our centre. The species isolated were E. avium, E. faecalis, and E. faecium. All the isolates demonstrated a high susceptibility to betalactams and vancomycin. The high rates of isolation could be due to the use of chromogenic media for primary culture of the pus and automated identification systems like the Vitek 2.
Beta haemolytic Streptococci (22), alpha haemolytic Streptococci (8), and S. pneumoniae (5) were mostly otogenic and were highly susceptible to penicillin and other betalactams. The isolation of these important pathogens was more in the study group II, often as single isolates.
Aerobic Gram-negative bacilli (62/193, 32.1%) though mainly otogenic, these organisms were also frequently isolated from the postcraniotomy and hematogenic abscesses. The enteric bacilli, such as Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Enterobacter species, Proteus species, Morganella morganii, and Providencia stuartii were the frequent isolates. Like the Gram-positive pathogens, the incidence of the Gram-negative bacilli, especially of multidrug-resistant and nosocomial pathogens, was seen to increase. E. coli and K. pneumoniae were highly resistant organisms producing extended-spectrum β-lactamase (ESBL). Nosocomial pathogens such as Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii) were also isolated.
5.1.2. Obligate Anaerobes (22 Single and 17 Mixed, Total 39)
An increasing incidence of polymicrobial anaerobic abscesses was documented. The spectrum of anaerobes in this study is shown in Table 2(b).
5.1.3. Uncommon Gram-Negative Bacilli
5.1.4. Mycobacterial Species (8/352, 2.2%)
All the 8 TB abscesses revealed AFB on microscopy, and M. tb was isolated by culture on LJ Medium (study group I) and BACTEC 460 TB system (Becton Dickinson, USA). In our study group I, two cases of mycobacterial abscesses, (M. tb (1) and M. fortuitum (1)  were documented, while there were 6 cases of tuberculous brain abscesses and one was a mixed fungal and TB abscess , in the study group II.
5.1.5. Nocardial Species (3/302, 1%)
There were 2 parietal lobe abscesses caused by N. asteroides in our group (one in a postrenal transplant patient and the other in a case of atopic dermatitis on steroids). We also reported a case of a young female with mycetoma on the back, who presented with an epidural abscess. N. brasiliensis was isolated from the sinuses on the skin and from the epidural abscess pus .
5.2. Fungal Agents
There were 5 (1.7%) cases of fungal brain abscess in our series, that occurred in various clinical settings. The cases with Aspergillus abscesses had a fatal outcome.
We reported a case of a 23-year-old immunocompetent male who sustained a head injury and developed a capsuloganglionic region abscess due to the neurotropic fungus, Cladophialophora bantiana . Diagnosis was made based on the microscopy, mycology, and histopathology findings of the aspirate obtained from the abscess. The patient responded clinically to amphotericin B.
Brain abscesses have been well known and reported from the beginning of the Hippocratic era [10, 11]. Essentially, a brain abscess is a focal intraparenchymal collection of pus and is classified based on the anatomical location or the etiologic agent causing it. It begins as a localized area of cerebritis and evolves into a collection of pus surrounded by a vascularized capsule [5, 20].
Our data shows that the incidence of brain abscess continues to be significant in the neurosurgical clinical setting. The average incidence of brain abscess among all the space-occupying lesions in our institute is about 25%. Recent studies from India have reported an average of 9 to 15 cases per year [1, 7, 8], while studies from developed countries recorded a lower number, 5–12 cases per year [10, 21, 22]. As per our data and other studies, brain abscesses occur in all decades of life, with a male preponderance [1, 6, 21, 22]. However, the reasons for the male preponderance, are not clear .
The spectrum of organisms seen in brain abscess usually depends on the primary source of infection. Gram-positive cocci, especially the Enterococci, and Gram-negative bacilli, especially E. coli and K. pneumoniae, were more frequently isolated in group II than in the earlier group I. This is probably due to the use of the Bact/alert system and direct inoculation of the purulent aspirates into the bottles. A larger number of antibiotic-resistant Gram-negative bacilli were isolated from cases in group II, probably due to the increased number of trauma cases in this group.
6.1. Otogenic Abscesses
CSOM continues to be the most frequent predisposing condition in all age groups (24/50 (48%) and 118/302 (39%) in both the groups), respectively, time groups. Other studies from India also documented CSOM as a major source of brain abscess, 49% , 31.4% , and 40% . Though middle ear suppurative disease was seen to extend to temporal lobe or cerebellum [1, 23, 24], multiple otogenic abscesses, often involving the frontal and parietal lobes, were seen in the study group II, probably due to arterial dissemination of infective emboli. Direct extension may also occur through osteomyelitis in the posterior wall of the frontal sinus, sphenoid, and ethmoid sinuses. This direct route of intracranial extension is more commonly associated with subacute and chronic otitic infection and mastoiditis than with sinusitis . Frontal or ethmoid sinus infections generally spread to the frontal lobes. Odontogenic infections can spread to the intracranial space via direct extension or a route, generally to the frontal lobe .
All the otogenic abscesses, in our study, were of bacterial etiology (aerobic and/or anaerobic), with culture positivity of 85%. Gram-positive facultative aerobes were the most frequent isolates from these abscesses, as also recorded in other studies [1, 6, 7, 23, 24]. An unusual case of concomitant TB (M. tb) and fungal (Fonsecaea pedrosoi) infections involving the middle ear cleft extending and destroying the craniovertebral junction at the skull base was successfully managed and treated at our centre . Our study and others from India clearly suggest that middle ear infections need to be treated aggressively to reduce the incidence of brain abscesses [1, 24].
6.2. Odontogenic Abscesses
The oral and dental florae, mainly from the subgingival sites, are documented to be frequent sources of brain abscesses . These sites are usually comprised of Streptococcus milleri and the Gram negative anaerobic bacilli. Two cases of odontogenic abscesses were noted in the study group II caused by S. aureus and Prevotella melaninogenica.
6.3. Cardiogenic Abscesses
Cyanotic congenital heart diseases such as tetralogy of Fallot (10/23, 43.5%), transposition of the great vessels (3/23, 13%), and dextrocardia (2/23, 0.08%) are documented as risk factors for brain abscess [1, 8, 26, 27] and congenital pulmonary arteriovenous malformations . We documented cardiogenic abscesses in 18% (9/50) in the study group I  and 7.6% (23/302) in the study group II. The age of the patients with cardiogenic abscess was less than 20 years though there was one 45-year-old patient with an S. aureus abscess.
Patients with cyanotic heart disease have a right to left shunt of venous blood in the heart, bypassing the pulmonary circulation, thus leading to bacteremia, septicemia, and infective thromboembolism, usually in the brain. These patients also have low-perfusion areas in the brain due to chronic hypoxemia and secondary polycythemia. The abscesses, often multiple, occur at the junction of the gray and white matter. The parietal lobes are most commonly affected due to the large caliber and direct continuation of the middle cerebral artery . The congenital arteriovenous anomalies become a nidus for the organisms, especially Gram-positive ones, spread to the brain leading to the development of brain abscess [1, 9]. Nineteen of the 23 (82.6%) cardiogenic abscesses in our series were positive for bacteria by microscopy and Gram-positive cocci, including facultative aerobes, and obligate anaerobes were isolated.
6.4. Traumatic Abscesses
Trauma to the skull, either following road traffic accident (21/302 7.2%) or a craniotomy (20/302 6.6%), is an important risk factor in the study group II, which was not encountered in the study group I, probably related to increase in speeding vehicles and availability of neurosurgical facilities. Thirty six of the forty one abscesses were solitary, and the parietal lobe was the most commonly affected site.
Microscopy revealed organisms in 92.7% abscesses, while 80.5 of them were culture positive. S. aureus infection was common in the RTAs, probably due to the direct implantation of the organism derived from the normal flora of the calvarial skin. On the other hand, the postcraniotomy abscesses had a spectrum that is predominantly of gram-negative facultative aerobes and was often a nosocomial infection. These isolates, most commonly E. coli and K. pneumoniae, had a high level of resistance to antibiotics. Strict aseptic measures during the postoperative management of the surgical wound and care of the intravascular catheters in these patients is very important and cannot be underscored [6, 11].
Postcraniotomy brain abscesses due to C. bantiana were reported by other centers [29–31]. The portal of entry of the fungus is either due to direct inoculation following trauma to the skull, either following RTA or craniotomy, inhalation of the spores, or a haematogenous spread to the brain .
6.5. Hematogenic Abscesses
Septicemia and/or sepsis elsewhere in the body is a probable cause for the spread of the organism from the primarily involved organ to the brain. We documented 10.9% hematogenic abscesses in the study group II. Twelve of the abscesses (36.4%) had no detectable organisms on microscopy and were sterile on culture. Menon et al. recorded 40% sterile abscesses in patients on prior antibiotics .
Also calling metastatic abscesses from a remote site, the hematogenic abscesses are the commonest type in developed countries . They are often multiple and typically occur at the junction of the white and gray matter, where the capillary blood flow is the slow . They are more commonly seen along the distribution of the middle cerebral arteries and the parietal lobes, where the regional blood flow is the highest. The common systemic sources of infection are chronic pulmonary infections, skin pustules, bacterial endocarditis, and osteomyelitis. Those with a right to left vascular shunt as a result of congenital heart disease or pulmonary arteriovenous malformations are particularly susceptible. Being hematogenous, any lobe of the brain can be affected, and some of them could be multiple [9, 28]. Though these abscesses are reported to contain a mixed flora , in the present series, the infected hematogenic brain abscesses were monomicrobial, since the septicemia is usually monomicrobial. Gram-negative pathogens were more frequently isolated from these abscesses, though unusual Gram-negative organisms such as S. typhi and B. pseudomallei were isolated from the hematogenic brain abscess pus.
6.6. Preexisting Pulmonary Lesions
Primary infections or secondarily infected pulmonary cavities can be a predisposing cause of brain abscess, especially in the immunocompromised patients . Six cases of brain abscesses of a probable pulmonary origin were recorded in group I which include 1 M.tb, 1 M. fortuitum, and 4 bacterial abscesses . In the study group II, there were 3 abscesses of proven pulmonary origin (organisms were isolated from the respiratory tract), which grew N. asteroides (a case of postrenal transplant) and M.tb (2 cases), respectively.
The urinary tract infections are an important primary source of brain abscess. We documented an abscess in lower midbrain in a patient with urosepsis. The abscess was sterile, probably due to prior antibiotic therapy. A similar report of brain abscess due to urosepsis was reported in literature .
6.8. Intracranial and Meningeal Lesions
Brain abscess has been documented to occur following a contiguous spread from infected foci within the brain or meninges or a secondary infection of preexisting intracranial lesions [4, 33]. Two brain abscesses from a primary infective focus within the brain or the meninges were documented in our study. One was a case of neurocysticercosis developing a temporal abscess due to Neisseria meningitides, probably a secondary infection of the cystic lesion. The other case was a case of pyogenic meningitis developing a frontoparietal abscess due to Peptostreptococcus species.
6.9. Osteomyelitis of the Calvarium
Osteomyelitis of the skull bones can be a primary focus with a contiguous spread to the underlying parenchyma  as noted in our study . We documented an unusual case of B. pseudomallei calvarial osteomyelitis that had spread to both the parietal lobes and medially extended up to the sagittal sinus . There were two other cases of calvarial osteomyelitis with sterile abscesses in the study group II.
Immunosuppression can predispose patients to the development of brain abscesses. With the increasing number of transplant surgeries, especially renal, in the recent times, it is a significant emerging problem . Immunosuppression induced by steroids also may predispose the patients in developing brain abscess.
The spectrum of organisms noted in the immunosuppressed patients may not be found in immunocompetent individuals, and because of this, empirical therapy in these patients should be avoided . Since, the imaging features of the abscess on computerized tomography or magnetic resonance imaging (MRI) also do not help in the diagnosis, attention should be directed to obtaining a microbiological diagnosis, whenever possible, so that appropriate antimicrobial therapy can be initiated without delay. The pus obtained from the abscess should be subjected to microbiological examination for fungal elements, AFB including Nocardia and parasites besides the routine aerobic and anaerobic cultures [1, 7, 9]. These compromised hosts with impaired T-lymphocyte or macrophage function are prone to develop infections with intracellular pathogens such as fungi (particularly Aspergillus species) and bacteria like Nocardia species [6, 11, 34], especially emerging Nocardial species (N. cyriacigeorgica) .
Brain abscess was documented in 3 cases of renal transplantation. The aspirated materials on microscopy, and culture revealed growth of A. flavus, N. asteroids, and S. pneumoniae along with S. aureus in the 3 cases, respectively (Table 2(c)). Another fatal case of mycotic brain abscess in a case of atopic dermatitis on immunosuppressive therapy with steroids in a 5-year-old female child and multiple parietal abscesses secondary to A. terreus and N. asteroides, probably originating from the ears or the lungs, were noted. Dias et al. documented a case of Nocardial brain abscess in an elderly male with undetected diabetes mellitus [34, 36].
6.11. Cryptogenic Brain Abscesses
This group comprises of those abscesses where there would be no obviously demonstrable primary focus of infection elsewhere in the body nor any underlying predisposing condition leading to infection. The reported incidence of such cryptogenic abscesses, as per several studies ranges between 15 and 22% [1, 10, 34]. The organisms in the cryptogenic abscesses have been shown to be derived from the upper respiratory tract and oral flora, comprising mainly of Streptococcal species and anaerobic cocci [1, 10]. In one of the studies, a patent foramen ovale was identified by echo cardiogram and was proposed as a possible way of migration and seeding of the oral flora to the brain .
We documented 23.3% brain abscesses as cryptogenic. Though the majority were solitary and sterile abscesses (53/82, 64.6%), the culture-positive cryptogenic abscesses (19/82, 26.4%) were caused mainly by a single Gram-positive facultative aerobe (12/19), among which S. aureus (MSSA) was the predominant isolate. The remaining cases were polymicrobial.
7. Microscopy versus Culture
The sensitivity of microscopy depends on the number of organisms in the specimen (10 3 CFU/mL). In our study, the Gram's stain was positive in 30 culture-negative cases, while it was negative in 12 culture-positive cases. There is a need to adopt methods to improve the detection rates especially by microscopy, which includes fluorescent staining using acridine orange (sensitivity (10 2 CFU/mL)) , calcoflour white for fungal filaments, and by auramine rhodamine fluorescent stain for mycobacteria.
8. Culture for Various Organisms
All the culture methods used in our study were to optimize the isolation of the various possible and cultivable etiologic agents. It has been shown that a direct inoculation and use of automated methods of culturing the pus, immediately after its aspiration, enhances the yield of organisms [7, 38]. Direct inoculation of the pus specimen into a standard anaerobic BacT/Alert bottle (bioMérieux, USA) facilitated early and better yield of the organisms. The unvented, bottled anaerobic medium facilitates the growth of both facultative aerobes and the obligate anaerobes, and the yield is further enhanced by the shaking incubator, inbuilt in the bacT/alert system .
Despite the meticulous conventional microbiological procedures, 6/50 (12%) and 99/302 (32.8%) abscesses were sterile, in our group, respectively. The reported incidence of sterile abscesses, from other centers, has ranged between 0% and 43% [1, 7, 39]. The yield of organisms by culture also directly depends on the prior use of antibiotics . This could be one of the reasons for an increased incidence of sterile abscesses in group II of our study.
Infections with P. aeruginosa and A. baumannii are caused by contiguous extension to brain and meninges from an ear, mastoid, paranasal sinus surgery, or diagnostic procedures. In some patients, the involvement of the CNS is due to spread of the organism from infective endocarditis, pneumonia, or urinary tract infection . These organisms have emerged as important and highly resistant nosocomial pathogens in recent years.
Brain abscess due to unusual Gram-negative bacilli was observed in the study group II only, probably due to the improved microbiological diagnostic techniques and automated identification systems used. (i) Salmonella typhi is a rare pathogen in brain abscess and probably spreads to the brain hematogenously . (ii) B. pseudomallei (agent of Melioidosis): brain abscess is a complication of a neurological infection . Isolation of B. pseudomallei from the specimen remains the “gold standard” in diagnosis. The microbiologist should be well versed with the colony morphology and the antibiogram of these important yet not very fastidious organisms. Ceftazidime remains the drug of choice in the early phase of treatment, followed by a prolonged therapy with cotrimoxazole .
Anaerobic brain abscesses: the earlier studies on brain abscesses from India were specifically on anaerobic infections [41–43]. The obligate anaerobes are significant pathogens of a brain abscess and often occur as mixed infection either with another anaerobe or a facultative aerobe, as seen in our group. They are generally associated with otogenic and odontogenic infections.
Anaerobes are highly susceptible to metronidazole . Subsequent to its addition to the antibiotic treatment regimen of a brain abscess and widespread use, the incidence of anaerobic infections has declined [1, 12, 41]. Attention to proper anaerobic isolation techniques is essential for a good recovery [11, 13] as is evidenced in the increased yield of anaerobes in our study group II (Table 2(c)). Since conventional anaerobic cultures require 2-3 days of incubation, it is essential to use alternative techniques for their early detection [44, 45]. Gas liquid chromatography (GLC) is a routine method used in the identification and differentiation of anaerobes from aerobes in clinical samples on the basis of the presence of volatile and nonvolatile fatty acids on the chromatogram. Computer-aided GLC is commercially available and is a means of rapid microbial identification [44, 45]. However, the equipment and cost are the limiting factors for a routine use of GLC in resource-restricted conditions.
Tuberculous brain abscess results when mycobacteria gain entry to the brain parenchyma, by a hematogenous route from a remote site, usually the lungs, unlike TB Meningitis, which occurs via lymphatic spread from cervical lymph nodes. The TB bacilli are immobilized in end arteries, which lead to formation of submeningeal tuberculous foci and either result in a tuberculoma or undergo a central caseation and liquefaction to form an abscess [46–49]. This phenomenon is very rare and commonly occurs in patients with cell-mediated immunity and is mostly focal and usually secondary to a primary focus in the lungs. Histologically and clinically, these abscesses are devoid of a granulomatous reaction, similar to pyogenic abscesses .
For appropriate therapy and clinical management, a TB abscess must be differentiated from a tuberculoma. The criteria for the diagnosis of a TB brain abscess, laid down by Whitener in 1978, should be fulfilled in the diagnosis of a TB brain abscess . These include (i) evidence of a true abscess formation within the brain, as confirmed during surgery, (ii) histological proof of the presence of inflammatory cells in the abscess wall (histologically, the abscess walls are usually devoid of epitheloid and giant cells, unlike in a tuberculoma , and (iii) demonstration of AFB and isolation of M.tb from the abscess pus. Though not included in this data, we had reported a case of calvarial tuberculous osteomyelitic abscess, which was successfully managed .
Nocardial brain abscesses are rare and account for about 1-2% of all cerebral abscesses [52–54]. The entity is being increasingly reported in the present era of transplant surgeries and immunosuppressive therapies. The infection may occur as an isolated lesion or as a part of a disseminated infection of a pulmonary or a cutaneous infection [6, 11, 16, 34, 52, 53, 55]. Almost all the patients with Nocardial brain abscesses have a defective cell-mediated immunity . An early detection and treatment are very important since the mortality is three times higher than that of other bacterial brain abscesses. Though N. asteroides and N. brasiliensis are the common species, other species are also being isolated from brain abscess . The diagnosis of Nocardial brain abscess requires a high index of clinical suspicion, with an attempt for an early tissue and microbiological diagnosis .
Invasive fungal infections remain a life-threatening complication in children with hematological malignancies. The brain is a common site of haematogenously disseminated fungal infections from an extracranial focus . Cerebral aspergillosis occurs in about 10 to 20% of all cases of invasive aspergillosis and has a very poor prognosis. The outcome depends on the early recognition of the causative organism and prompt initiation of antifungal treatment. Hence, every attempt should be made to detect fungal filaments in the brain abscess material submitted .
9. Alternative and Advanced Techniques to Improve Detection of the Infecting Pathogen(s)
Microscopy-positive but culture-negative abscesses, especially the bacterial, are not unusual and pose a challenge to the microbiologist(s). The organisms, often the nutritionally demanding Streptococcus species and the anaerobic cocci, generally will not be recovered on routine culture media. Also cultures will be negative when the number of organisms in the abscess pus is less. Such specimen should ideally be processed for the etiologic agent by more sensitive assays. At present, these highly sensitive and sophisticated assays are being increasingly used in advanced centers for research purposes. (1) Broad range real-time polymerase chain reaction (RT PCR) using 16srRNA or DNA will help amplify and detect the probable bacterial nucleic acid, as shown by several studies [57–62]. A subsequent sequencing of the amplified product is performed to further improve the specificity. With the recent advances, the molecular methods are gradually making inroads into routine diagnostic laboratories and in the coming years may become important primary tools in the early detection of the etiologic agent. However, the specificity and the cost of these assays are important limiting factors  in the underdeveloped and developing countries. (2) In vitro nuclear magnetic resonance (NMR) used for detecting the specific spectral pattern of the amino acids and other volatile substances released by metabolic processes of the bacteria has been applied as a means of differentiating abscesses from other space-occupying lesions in the brain [64–66]. However, the need for NMR with very high Tesla and an expert analysis and interpretation of the spectral patterns are the limiting factors. (3) Gas liquid chromatography (GLC) on the pus sample is also an alternative method, especially for the anaerobic abscesses.
As evidenced by our data, the microbiology of intracranial abscesses is complex. The detection and identification of the causative pathogen(s) are the cornerstones of diagnosis for an appropriate management and therapeutic optimization. The value of a promptly evaluated microscopy coupled with meticulous and elaborate culture methods of the abscess material cannot be under-estimated. The high yield of positive cultures will then enable the neurosurgeon and the infectious disease specialist to treat brain abscess more rationally and appropriately. Abscesses rarely arise de novo within the brain [4, 11]. There is almost always a primary lesion elsewhere in the body that must be sought assiduously, since failure to treat the primary lesion will result in relapse . The need to sample the primary source of the brain abscess, especially lungs, when unusual pathogens are isolated from the brain abscess (even in cryptogenic abscesses), is emphasized through our series.
It is advisable for the neurosurgeon to co-ordinate closely with the microbiologist and ensure application of advanced microbiological and molecular techniques, to detect the new and emerging agents of intracranial abscesses.
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3. Results and Discussion
3.1. Molecular and Morphological Identification of Bacterial Endophytes from C. macowanii leaves
Endophytes inhabit unique biological niches growing in unusual environments. Their isolation and identification are vital for further exploration [49, 50]. In this study, a total of 9 bacterial endophytes were isolated and characterized from the leaves of C. macowanii. as seen in Table 1 .
Seven Gram-negative bacteria were observed. Phenotype diversity of the endophyte contributes to the Gram reaction and colony morphology of the endophytes  and hence the diverse endosphere, while the growth rate and size of the host plant also have an effect on the diverse endophytic community . The endophytic community of plants is influenced by the age of the host plant, geographic location, and abiotic factors such as temperature [52, 53]. This would explain the diversity of the bacterial endophytes isolated.
Bacterial genera including Pseudomonas, Bacillus, and Burkholderia have been isolated as endophytes from leaves of medicinal plants . These bacteria genera predominate in medicinal plants, as they assist the host plant in mineral nutrient composition and protection against abiotic and biotic stresses . The BLAST search conducted of the results of the 16S rDNA gene sequence revealed that the isolated endophytes belong to bacterial genera, such as Raoultella, Acinetobacter, Pseudomonas, Bacillus, Enterobacter, and Arthrobacter, as seen in Figure 1 . This supports our observed results.
Neighbor-joining phylogenetic tree of 16S rRNA gene sequences of endophytes isolated from C. macowanii leaves showing the relationship with other similar species selected from GenBank.
Pseudomonas are common bacteria associated with plants and have been isolated from a number of plant species and tissues. They display a positive effect on host plant growth such as reducing drought stress and producing plant hormones such as 1-aminocyclopropane-1-carboxylic acid (ACC) and Indole-3-acetic acid (IAA) and acting as biocontrol agents [26, 49, 56, 57]. Our isolate TES05A showed 94% similarity with Pseudomonas sp. WB4.4-99. Endophytic isolate TES14A showed 83% similarity with Pseudomonas cichorii Pc-Gd-1. Endophytic Pseudomonas cichorii has been isolated from potato cultivar . Isolate TES06A displayed a 99% similarity with Pseudomonas putida PF45. Endophytic Pseudomonas putida have been isolated from mango orchard . Isolate TES05B showed 99% similarity with Pseudomonas palleroniana IHB B 7234. Endophyte Pseudomonas palleroniana has been isolated from bananas and is reported to fix free nitrogen, solubilize phosphates, and produce siderophores in vitro .
TES02C displayed a sequence identity of 100% to Acinetobacter guillouiae OTU-b62. Different Acinetobacter species have been isolated from potato cultivars . TES15A isolates revealed 100% to Arthrobacter pascens H19. Arthrobacter spp. has been isolated from ethnomedicinal plants in Southern India . Bacillus endophytes have been isolated from sunflower, potatoes, and cotton and assist host plants in phosphate solubilization and auxin production . Our endophytic isolates (TES07A and TES07B) displayed 100% similarity to Bacillus safensis TMV13-3. Bacillus spp. Isolates TES02B had a similarity of 62% to Raoultella terrigena m 5. Endophytic Raoultella ornithinolytica has been isolated from mountain-cultivated ginseng plants . A 64% similarity was observed between our isolate TES10A and Enterobacter asburiae E6 – 2. Endophytic Enterobacter asburiae has been isolated from date palm and promotes plant growth .
To the best of our knowledge, this is the first report on the isolation of Arthrobacter pascens and Enterobacter asburiae from C. macowanii.
3.2. Antibacterial Evaluation of Crude Bacteria Endophyte Extracts from the Leaves
The lowest MIC (0.0625 mg/mL) was observed from Arthrobacter pascens crude extract and crude extracts against B. subtilis, respectively. The crude extracts of most of the endophytes showed MIC values below 1.00 mg/mL. The leave crude extracts displayed noteworthy activity against both Gram-positive and Gram-negative bacteria as seen in Table 4 .
Antibacterial evaluation of C. macowanii crude leave extract and crude endophyte extracts from the leaves.
|Test organism with MIC (mg/mL)|
|Crude extracts||B. cereus||B. subtilis||S. epidermidis||S. aureus||M. smegmatis||M. marinum||E. aerogenes||E. coli||K. pneumonia||P. vulgaris||P. aeruginosa|
|Positive control MIC (μg/mL)|
C. macowanii leaves have been used traditionally to cleanse the blood, treat coughs, kidney, and bladder diseases in humans and animals, and also t treat coughs and diarrhea . The results obtained in this study indicate the inhibition of B. subtilis, M. smegmatis, and P. vulgaris at 0.500 mg/mL, and S. aureus, E. coli, and K. pneumonia were inhibited at 0.250 mg/mL and S. epidermidis and P. aeruginosa at 1.00 mg/mL and 0.125 mg/mL, and respectively.
These data provide scientific justification for the ethnomedicinal uses of the leaves, as the inhibited bacteria are the main causative agents of the ailments the leaves are used to treat. S. aureus, E. coli, and K. pneumoniae were inhibited by a final concentration of 250 μg·mL 𢄡 by alkaloids such as crinine, cherylline, crinamidine, 3-O-acetylhamayne, and bulbispermine which have been isolated from C. macowanii leaves . To the best of our knowledge, this study is the first to report on the extraction of crude extract from leaves of C. macowanii and its antibacterial activity.
The cultivation of plants to obtain bioactive compounds has led to drawbacks, such as overharvesting of plants to obtain bioactive compounds, different environmental conditions tend to produce low yields, and total synthesis and semisynthesis are challenging due to complex structures . A number of endophytic microorganisms have produced anticancer, antimicrobial, antidiabetic, insecticidal, and immunosuppressive compounds . Plants growing in a variety of places possibly harbor endophytes with novel natural products [66, 67].
Raoultella ornithinolytica crude extract had MIC values ranging from 0.250 to 16 mg/mL, with the most significant inhibition observed for K. pneumonia, E. coli, and M. marinum at concentrations of 0.500 mg/mL, and P. aeruginosa was inhibited at concentrations of 0.250 mg/mL. Microcin genes have been reported to be present on Raoultella ornithinolytica  and microcins are antibacterial peptides produced by Enterobacteria . This could explain the observed results. Acinetobacter guillouiae crude extract showed MIC values of between 0.500 and 16 mg/mL. The crude extract showed activity against M. marinum at 0.500 mg/mL.
Bacillus safensis crude extract showed MIC values of between 0.125 and 㸖 mg/mL. The crude extract showed activity against B. subtilis, M. marinum, and E. coli at concentrations of 0.125 mg/mL, 0.500 mg/mL, and 0.250 mg/mL, respectively. Crude endophytic extracts of Bacillus safensis isolated from Ophioglossum reticulatum L. displayed antibacterial activity against Staphylococcus aureus and Escherichia coli . This is in agreement with the obtained results.
Enterobacter asburiae crude extract showed MIC values of between 0.125 and 16 mg/mL. The crude extract showed activity against M. smegmatis and E. coli at concentrations of 0.500 mg/mL and M. marinum and K. pneumonia at concentrations of 0.125 mg/mL. Endophytic crude extracts of Enterobacter asburiae displayed antibacterial activity against K. pneumoniae, E. coli, S. aureus, and B. cereus. Enterobacter strains  have been reported to produce antibacterial lipopeptides with a broad activity . This supports the obtained results.
Arthrobacter pascens crude extract showed MIC values of between 0.0625 and 㸖 mg/mL. The most active inhibition was against B. subtilis at 0.0625 mg/mL. The crude extract showed activity against S. aureus and E. aerogenes at concentrations of 1.00 mg/mL. Arthrobacilin, an antibacterial compound produced by Arthrobacter spp., showed inhibition against S. aureus [72, 73]. This could explain the antibacterial activity observed.
Pseudomonas sp. crude extract showed MIC values of between 0.0625 and 㸖 mg/mL. The most active inhibition was against M. marinum at 0.0625 mg/mL. Mupirocin produced by Pseudomonas strains has been reported to possess antibacterial activity . Pseudomonas palleroniana crude extract showed MIC values of between 0.250 and 㸖 mg/mL. The most active inhibition was against P. vulgaris at 0.250 mg/mL. The crude extract showed activity against E. aerogenes and P. aeruginosa at concentrations of 1.00 mg/mL and M. marinum and K. pneumonia at concentrations of 0.500 mg/mL. Endophytic crude extracts from Pseudomonas palleroniana were reported to inhibit Escherichia coli and Staphylococcus aureus . Pyoluteorin produced by Pseudomonas palleroniana strains has been reported to contain antibacterial activity . Pseudomonas putida crude extract showed MIC values of between 1.00 and 㸖 mg/mL. The most active inhibition was against P. aeruginosa at 1.00 mg/mL. Antibiotics pyoluteorin, phenazine-1-carboxamide, and phenazine-1-carboxylic acid were produced by Pseudomonas putida strains . This could explain the antibacterial activity observed. Pseudomonas cichorii crude extract showed MIC values of between 0.125 and 16 mg/mL. The crude extract showed activity against E. coli and P. vulgaris at concentrations of 1.00 mg/mL, M. smegmatis and P. aeruginosa at concentrations of 0.125 mg/mL, B. subtilis at 0.250 mg/mL, and K. pneumonia at 0.500 mg/mL. To the best of our knowledge, this is the first report on the antibacterial activity of crude endophyte extracts from Pseudomonas cichorii.
Cos et al.  state that a concentration of π.1 mg/mL for a crude sample is the ideal concentration for anti-infective bioassays whereas [77, 78] recommend that crude samples with a concentration of 1.00 mg/mL and μg/ml (0.100 mg/ml) and μg/ml are considered to be very significant and moderately significant and therefore noteworthy for minimal inhibitory concentration. Stringent endpoints for anti-infective bioassays ought to be set to prevent false results and confusion, taking into consideration the sensitivity of extracts and test microorganisms, extraction methods, and solvents used [66, 67].
It was observed from the results that crude endophyte extract from Pseudomonas sp. and Arthrobacter pascens obtained from C. macowanii leaves had noteworthy antibacterial activity against the pathogenic bacteria used in this study and can be used as antibacterial agents against and M. marinum and B. subtilis infections, respectively.
3.3. Anticancer Evaluation of Crude Bacteria Endophyte Extracts from the Leaves against Resistance Cancer Cell Lines
Secondary metabolites produced by endophytic microorganisms' act as anticancer agents and display significant potential in medical and veterinary treatments [79, 80]. Anticancer agents paclitaxel and podophyllotoxin have been isolated from endophytic microorganisms .
3.3.1. Anticancer Evaluation of Crude Bacteria Endophyte Extracts from the Leaves against A549 Lung Carcinoma Cells
Pseudomonas putida and Bacillus safensis crude extracts showed a 47% and 50% cell reduction, respectively, against lung carcinoma cells at a concentration of 100 μg/mL as seen in Figure 2 .
Cytotoxic activity of endophytic-derived secondary metabolites and crude extracts on A549 lung carcinoma cells tested at different concentrations ranging from 100 to 3.13 μg/mL. The positive control used was auranofin. T1 = C. macowanii leaves, T2 = Raoultella ornithinolytica, T3 = Acinetobacter guillouiae, T4 = Pseudomonas sp., T5 = Pseudomonas palleroniana, T6 = Pseudomonas putida, T7 = Bacillus safensis, T8 = Enterobacter asburiae, T9 = Pseudomonas cichorii, T10 = Arthrobacter pascens.
3.3.2. Anticancer Evaluation of Crude Bacteria Endophyte Extracts from the Leaves against UMG87 Glioblastoma Cells
Acinetobacter guillouiae crude extracts showed a 42% reduction of UMG87 glioblastoma cells at a concentration of 6.25 μg/mL and Arthrobacter pascens crude extracts displayed cell reduction of 37% at a concentration of 12.5 μg/ml as seen in Figure 3 .
Cytotoxic activity of endophytic-derived secondary metabolites and crude extracts on UMG87 glioblastoma cells tested at different concentrations ranging from 100 to 3.13 μg/mL. The positive control used was auranofin. T1 = C. macowanii leaves, T2 = Raoultella ornithinolytica, T3 = Acinetobacter guillouiae, T4 = Pseudomonas sp., T5 = Pseudomonas palleroniana, T6 = Pseudomonas putida, T7 = Bacillus safensis, T8 = Enterobacter asburiae, T9 = Pseudomonas cichorii, T10 = Arthrobacter pascens.
To the best of our knowledge, this is the first report on the anticancer activity of C. macowanii leave crude extracts. No noteworthy activities were observed from the leaves' crude samples against both cell lines used in this study. Bioactive compounds such as lycorine, pretazettine, crinamine, augustine, and galanthamine are noted to appear in C. macowanii leaves and have been reported to possess anticancer activity .
To the best of our knowledge, this is the first report on the anticancer activity of crude endophytes extracts from Pseudomonas palleroniana, Bacillus safensis, Enterobacter asburiae, Arthrobacter pascens, and Pseudomonas cichorii.
Acinetobacter guillouiae crude endophyte extract was the only tested sample that exhibited anticancer against UMG87 glioblastoma cells, with a 31% cell reduction at 100 μg/mL and 53% cell reduction at 3.13 μg/mL posing as a possible anticancer agent against brain cancer.
Bacillus safensis crude extracts displayed noteworthy activity against A549 lung carcinoma cells with 50% cell reduction at 100 μg/mL. Crude extracts of Bacillus safensis isolated from sea sponges had anticancer activity against HepG2 (hepatocellular carcinoma), HCT (colon carcinoma), and MCF 7 (breast carcinoma) . This could explain the observed results, and crude endophyte extracts from Bacillus safensis can be used as an anticancer agent against lung cancer.
Crude endophytic extract of Enterobacter asburiae, Pseudomonas sp., Arthrobacter pascens, and Pseudomonas palleroniana displayed no noteworthy activity against UMG87 glioblastoma cells and A549 lung carcinoma cells. The extracts can be tested on other cancer cell lines to determine their activity. Pseudomonas sp. are known to produce anticancer compounds and have been reported to have activity against a number of human cancer cell lines [86, 87]. Pseudomonas sp. and Pseudomonas cichorii displayed no noteworthy activity against UMG87 glioblastoma cells and A549 lung carcinoma cells. Pseudomonas palleroniana crude extracts displayed 36% cell reduction at 100 μg/mL against A549 lung carcinoma cells.
Crude endophyte extracts from Pseudomonas putida displayed 47% cell reduction at 100 μg/mL against A549 lung carcinoma cells. P. putida TJ151 is able to produce fluorouracil which is a bioactive aromatic compound and it is an anticancer drug . L-methioninase, an enzyme produced by Pseudomonas putida, has shown anticancer activity against leukemia cell lines, liver HepG2, breast MCF-7, lung A549, prostate PC3, and colon HCT116 [88, 89]. Methioninase from P. putida and 5-fluorouracil work synergistically to inhibit tumor growth and hence the activity observed .
Crude endophyte extracts from Raoultella ornithinolytica displayed 43% cell reduction at 100 μg/mL against A549 lung carcinoma cells. Protein complex from R. ornithinolytica has shown anticancer activity against HeLa cell line, human endometrioid ovarian cancer line (TOV 112D ATCC CRL-11731), and the human breast adenocarcinoma line (T47D ECACC 85102201) resulting in cytopathic effect and reduction in the cell number [91, 92].
Crude endophyte extracts of Raoultella ornithinolytica, Pseudomonas palleroniana, Pseudomonas putida, and Bacillus safensis can be further purified and tested for their anticancer activity against other types of the cancer cell line.
3.4. Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry LC-Q-TOF-MS Analysis
The isolation and characterization of bioactive secondary metabolites help in distinguishing between new and already known bioactive secondary metabolites which help in the development and discovery of new drug leads . Lycorine and powelline were some of the identified secondary metabolites present in the crude extract of C. macowanii leaves, Bacillus safensis, Pseudomonas cichorii, and Arthrobacter pascens. These are indicated in Table 5 .
LC-Q-TOF-MS analysis of crude extracts of C. macowanii leaves and their bacterial endophytes.
|Compound name||Rt (min/sec)||m/z||Molecular formula||Reported biological activity||Sample|
|Melicopicine||5.64||330.1351||C18H19N1O5||Antiplasmodial activity ||T1|
|Lycorine||1.92 13.76 36.75 7.23||288.1236||C16H17N1O4||Antibacterial cytotoxic and antitumor activities ||T1, T7, T9, T10|
|Angustine||10.35 19.23 25.98 11.07||314.1388||C20H15N3O1||Antiproliferative activity ||T1, T7, T9, T10|
|3-O-methyl epimacowine||5.63||288.1586||C17H21NO3||Anticancer ||T7|
|Crinamidine||17.57 15.50 15.44 6.32||318.1348||C17H19N1O5||Antibacterial  anticancer activity ||T1, T7, T9, T10|
|Vasicinol||4.18 21.69 21.51 8.91||205.0977||C11H12N2O2||Antibacterial activity ||T1, T7, T9, T10|
|Aulicine||1.99||320.1870||C18H25NO4||Anticancer activity ||T9|
|Powelline||19.14, 14.05, 17.98, 7.43||302.1389||C17H19N1O4||Antitumor  antibacterial ||T1, T7, T9, T10|
|Brefeldin A||11.52||281.1702||C16H24O4||Anticancer  and antifungal ||T10|
Not much work has been done to the leaves, and to the best of our knowledge, this is the first report of the identification of secondary metabolites from leaves of C. macowanii and their endophytes using LC-Q-TOF-MS. Endophytes are able to produce similar secondary metabolites as the host plants by exchanging fragments of their genomic DNA with the host plant [103, 104]. These secondary metabolites perform distinct functions such as antibacterial and anticancer activity as performed in this study . Melicopicine is an acridone alkaloid isolated from leaves of Teclea and Zanthoxylum species . An acridone alkaloid melicopicine has been isolated from Melicope fareana  and has anthelmintic and antibacterial activities . This would explain the noteworthy antibacterial activity of the crude leave extract observed in this study.
Lycorine is an alkaloid previously isolated from C. macowanii . Leaves of C. macowanii contain more lycorine as compared to the bulbs and other plant parts such as roots and flowers . Crude endophyte extras of Bacillus safensis, Pseudomonas cichorii, and Arthrobacter pascens displayed the presence of lycorine, and this is not surprising as endophytes can metabolize secondary metabolites from the host plant . Lycorine has been reported to possess antibacterial activity and cytotoxic and antitumor activities . This would explain the observed antibacterial and/or anticancer activity of the crude endophyte extracts.
Angustine is an alkaloid previously isolated from plants of the Rubiaceae and Loganiaceae family . To the best of our knowledge, angustine is being identified for the first time in C. macowanii leaves extracts and its isolated endophytes.
Aulicine and 3-O-methyl-epimacowine are crinine-type alkaloids and have been isolated from Hippeastrum aulicum Herb. and Hippeastrum calyptratum Herb. [110, 111]. Evidente and Kornienko  reported on the anticancer properties of these crinine-type alkaloids. This would justify the anti-lung cancer activity of crude Bacillus safensis endophyte extracts. Different cancer cell lines can be used to determine the anticancer activity of crude Pseudomonas cichorii endophyte extracts.
Crinine-type alkaloid crinamidine has been isolated from different plants of the Amaryllidaceae family  and also from the bulbs, flowering stalks, leaves, and roots of Crinum macowanii . Crinamidine is found in whole plant parts of the Crinum species . Powelline is an alkaloid reported to occur in C. macowanii  and Ndhlala et al.  reported its occurrence from the bulbs. Both these crinine-type alkaloids have been reported to possess antibacterial, antitumor, and anticancer activity [20, 98, 100]. This is not alarming as endophytes can metabolize secondary compounds from the host  and display a number of biological activities as the host plant.
Vasicinol is a quinazoline alkaloid from Adhatoda zeylanica Medic. . Crude endophyte extracts of Bacillus safensis, Pseudomonas cichorii, and Arthrobacter pascens displayed the presence of this alkaloid this is not surprising as some quinazoline alkaloids are produced by microbes . Vasicinol can be tested on other resistant pathogenic bacteria to combat antimicrobial resistance, as its antibacterial activity has been reported by Jain et al. .
Brefeldin A is a fungal metabolite produced by species of the Ascomycetes , and to the best of our knowledge, it is being reported for the first time in bacterial endophytes. Brefeldin A has been reported to possess anticancer activity  different cancer cell lines can be used to determine its anticancer activity with different cell lines.
From the results obtained, varying retention times were observed between the leaves and bacterial endophytes samples, even though the same chromatography conditions were used. Factors such as the affinity of the compounds to the extraction solvents used , the change in polarity of the sample being analyzed, and fragments that make up the secondary metabolites detected , and the formation of secondary metabolites complexes with extraction solvents used to influence the different retention times observed, as they create a sample matrix .
The identified alkaloids lycorine, crinamidine, and powelline are true alkaloids of the Amaryllidaceae family [120, 124], and this supports the obtained results as Crinum macowanii belongs to this plant family. The availability of these alkaloids leads to overuse and overharvesting of C. macowanii to obtain these bioactive compounds [65, 125]. The bioprospecting of endophyte isolated metabolites could help save the environment since endophytes have been reported to contain similar bioactive compounds as the host plant [66, 126] and in some doing medicinal plants are being conserved and revenue is generated by the bioprospecting of metabolites from endophytes .
Labilibaculum antarcticum sp. nov., a novel facultative anaerobic, psychrotorelant bacterium isolated from marine sediment of Antarctica
A novel facultative anaerobic and facultative psychrophilic bacterium, designated SPP2 T , was isolated from an Antarctic marine sediment. Cells of the isolate were observed to be long rods (0.5 × 5–10 μm), Gram-stain negative and to have gliding motility. For growth, the optimum NaCl concentration was found to be 2–3% and the optimum temperature to be 18–22 °C. Strain SPP2 T cannot use sulfate and nitrate as electron acceptors in the presence of lactate. The G+C content of the genomic DNA was determined to be 36.0 mol%.. The major cellular fatty acids were identified as anteiso-C15:0 and iso-C15:0. MK-7 was found to be the predominant respiratory quinone. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belongs to the family Marinifilaceae and to be closely related to Labilibaculum manganireducens 59.10-2M T with 16S rRNA gene sequence identity of 98%. The OrthoANI and dDDH values between the genome sequences of strain SPP2 T and its close relative were 84% and 27.3%, which are lower than the threshold values for species delineation. On the basis of phylogenetic and phenotypic characterisation, Labilibaculum antarcticum sp. nov. is proposed with the type strain SPP2 T (= NBRC 111151 T = CECT 9460 T ).
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The antimicrobial lipopeptides polymyxin B and E (colistin) are being used as a ‘last-line’ therapy for infections caused by multidrug-resistant Gram-negative pathogens. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections caused by the Gram-negative ‘superbugs’. This report details the structure–activity relationships (SAR) based design, in toto synthesis, and preclinical evaluation of a series of novel polymyxin lipopeptides with better antibacterial activity against polymyxin-resistant Gram-negative bacteria.
15.1.1: Gram Positive Rods - Biology
Gilbert Van Stappen
Laboratory of Aquaculture & Artemia Reference Center
University of Gent, Belgium
4.2.1. Cyst biology
220.127.116.11. Cyst morphology
A schematic diagram of the ultrastructure of an Artemia cyst is given in Fig. 4.2.1.
Figure 4.2.1. Schematic diagram of the ultrastructure of an Artemia cyst. (modified from Morris and Afzelius, 1967)
The cyst shell consists of three layers:
· alveolar layer: a hard layer consisting of lipoproteins impregnated with chitin and haematin the haematin concentration determines the color of the shell, i.e. from pale to dark brown. Its main function is to provide protection for the embryo against mechanical disruption and UV radiation. This layer can be completely removed (dissolved) by oxidation treatment with hypochlorite (= cyst decapsulation, see 4.2.3.).
· outer cuticular membrane: protects the embryo from penetration by molecules larger than the CO 2 molecule (= multilayer membrane with very special filter function acts as a permeability barrier).
· embryonic cuticle: a transparent and highly elastic layer separated from the embryo by the inner cuticular membrane (develops into the hatching membrane during hatching incubation).
The embryo is an undifferentiated gastrula which is ametabolic at water levels below 10% it can be stored for long periods without losing its viability. The viability is affected when water levels are higher than 10% (start of metabolic activity) and when cysts are exposed to oxygen i.e. in the presence of oxygen cosmic radiation results in the formation of free radicals which destroy specific enzymatic systems in the ametabolic Artemia cysts.
18.104.22.168. Physiology of the hatching process
The development of an Artemia cyst from incubation in the hatching medium till nauplius release is shown in Fig. 4.2.2.
Figure 4.2.2. Development of an Artemia cyst from incubation in seawater until nauplius release.
When incubated in seawater the biconcave cyst swells up and becomes spherical within 1 to 2 h. After 12 to 20 h hydration, the cyst shell (including the outer cuticular membrane) bursts (= breaking stage) and the embryo surrounded by the hatching membrane becomes visible. The embryo then leaves the shell completely and hangs underneath the empty shell (the hatching membrane may still be attached to the shell). Through the transparent hatching membrane one can follow the differentiation of the pre-nauplius into the instar I nauplius which starts to move its appendages. Shortly thereafter the hatching membrane breaks open (= hatching) and the free-swimming larva (head first) is born.
Dry cysts are very hygroscopic and take up water at a fast rate i . e . within the first hours the volume of the hydrated embryo increases to a maximum of 140% water content Fig. 4.2.3. However, the active metabolism starts from a 60% water content onwards, provided environmental conditions are favourable (see further).
The aerobic metabolism in the cyst embryo assures the conversion of the carbohydrate reserve trehalose into glycogen (as an energy source) and glycerol.
Figure 4.2.3. Cellular metabolism in Artemia cysts in function of hydration level.
Increased levels of the latter hygroscopic compound result in further water uptake by the embryo. Consequently, the osmotic pressure inside the outer cuticular membrane builds up continuously until a critical level is reached, which results in the breaking of the cyst envelope, at which moment all the glycerol produced is released in the hatching medium. In other words the metabolism in Artemia cysts prior to the breaking is a trehalose-glycerol hyperosmotic regulatory system. This means that as salinity levels in the incubation medium increase, higher concentrations of glycerol need to be built up in order to reach the critical difference in osmotic pressure which will result in the shell bursting, and less energy reserves will thus be left in the nauplius.
After breaking the embryo is in direct contact with the external medium through the hatching membrane. An efficient ionic osmoregulatory system is now in effect, which can cope with a big range of salinities, and the embryo differentiates into a moving nauplius larva. A hatching enzyme, secreted in the head region of the nauplius, weakens the hatching membrane and enables the nauplius to liberate itself into the hatching medium.
22.214.171.124. Effect of environmental conditions on cyst metabolism
Dry cysts (water content from 2 to 5% see worksheet 4.2.1. for determination of water content and Table 4.2.6. for practical example) are very resistent to extreme temperatures hatching viability not being affected in the temperature range -273°C to +60°C and above 60°C and up to 90°C only short exposures being tolerated.
Hydrated cysts have far more specific tolerances with mortalities occurring below -18°C and above +40°C a reversible interruption of the metabolism (= viability not affected) occurring between -18°C and +4°C and between ± 33°C and ± 40°C, with the upper and lower temperature limits vary slightly from strain to strain. The active cyst metabolism is situated between +4°C and 䕅°C the hatching percentage remains constant but the nauplii hatch earlier as the temperature is higher.
As for other environmental conditions, optimal hatching outputs are reached in the pH range 8-8.5. As a consequence, the addition of NaHCO 3 , up to 2 g.l -1 , to artificial or diluted seawater or to dense suspensions of cysts results in improved hatching. This might be related to the optimal pH activity range for the hatching enzyme.
An increased hatching has been reported with increasing oxygen level in the range 0.6 and 2 ppm, and maximal hatching obtained above this concentration. To avoid oxygen gradients during hatching it is obvious that a good homogeneous mixing of the cysts in the incubation medium is required.
As stated above, hatching in a higher salinity medium will consume more of the energy reserves of the embryo. Above a threshold salinity (varying from strain to strain, 䕾 g.l -1 for most strains) insufficient quantities of water can be taken up to support the embryos metabolism. Optimal salinity for hatching is equally strain-specific, but generally situated in the range 15-70 g.l -1 .
Although the physiological role of light during the hatching process is poorly understood, brine shrimp cysts, when hydrated and in aerobic conditions, need a minimal light triggering for the onset of the hatching process, related to light intensity and/or exposure time.
As a result of the metabolic characteristics of hydrated cysts, a number of recommendations can be formulated with regard to their use. When cysts (both decapsulated and non-decapsulated) are stored for a long time, some precautions have to be taken in order to maintain maximal energy content and hatchability. Hatchability of cysts is largely determined by the conditions and techniques applied for harvesting, cleaning, drying and storing of the cyst material. The impact of most of these processes can be related to effects of dehydration or combined dehydration/hydration. For diapausing cysts, these factors may also interfere with the diapause induction/termination process, but for quiescent cysts, uncontrolled dehydration and hydration result in a significant drop of the viability of the embryos.
Hatching quality in stored cysts is slowly decreasing when cysts contain water levels from 10 to 35% H 2 O. This process may however be retarded when the cysts are stored at freezing temperatures. The exact optimal water level within the cyst (around 5%) is not known, although there are indications that a too severe dehydration (down to 1-2%) results in a drop in viability.
Water levels in the range 30-65% initiate metabolic activities, eventually reducing the energy contents down to levels insufficient to reach the state of emergence under optimal hatching conditions. A depletion of the energy reserves is furthermore attained when the cysts undergo subsequent dehydration/hydration cycles. Long-term storage of such material may result in a substantial decrease of the hatching outcome. Cysts exposed for too long a period to water levels exceeding 65% will have completed their pre-emergence embryonic development subsequent dehydration of these cysts will in the worst case result in the killing of the differentiated embryos.
Sufficiently dehydrated cysts only keep their viability when stored under vacuum or in nitrogen the presence of oxygen results in a substantial depletion of the hatching output through the formation of highly detrimental free radicals. Even properly packed cysts should be preferentially kept at low temperatures. However, when frozen, the cysts should be acclimated for one week at room temperature before hatching.
As Artemia is an inhabitant of biotopes characterized by unstable environmental conditions, its survival during periods of extreme conditions (i.e. desiccation, extreme temperatures, high salinities) is ensured by the production of dormant embryos. Artemia females can indeed easily switch from live nauplii production (ovoviviparity) to cyst formation (oviparity) as a fast response to fluctuating circumstances. Although the basic mechanisms involved in this switch are not yet fully understood, but sudden fluctuations seem to trigger oviparity (oxygen stress, salinity changes. ). The triggering mechanism for the induction of the state of diapause is however not yet known. In principle, Artemia embryos released as cysts in the medium are in diapause and will not resume their development, even under favourable conditions, until they undergo some diapause deactivating environmental process at this stage, the metabolic standstill is regulated by internal mechanisms and it can not be distinguished from a non-living embryo. Upon the interruption of diapause, cysts enter the stage of quiescence, meaning that metabolic activity can be resumed at the moment they are brought in favorable hatching conditions, eventually resulting in hatching: in this phase the metabolic arrest is uniquely dependent of external factors (Fig. 4.2.4.). As a result, synchronous hatching occurs, resulting in a fast start and consequent development of the population shortly after the re-establishment of favorable environmental conditions. This allows effective colonization in temporal biotopes.
For the user of Artemia cysts several techniques have proven successful in terminating diapause. It is important to note here that the sensitivity of Artemia cysts to these techniques shows strain- or even batch-specificity, hence the difficulty to predict the effect on hatching outcome. When working with new or relatively unknown strains, the relative success or failure of certain methods has to be found out empirically.
In many cases the removal of cyst water is an efficient way to terminate the state of diapause. This can be achieved by drying the cysts at temperatures not exceeding 35-40°C or by suspending the cysts in a saturated NaCl brine solution (300 g.l -1 ). As some form of dehydration is part of most processing and/or storage procedures, diapause termination does not require any particular extra manipulation. Nevertheless, with some strains of Artemia cysts the usual cyst processing techniques does not yield a sufficiently high hatching quality, indicating that a more specific diapause deactivation method is necessary.
Figure 4.2.4. Schematic diagram explaining the specific terminology used in relation with dormancy of Artemia embryos.
Table 4.2.1. Effect of cold storage at different temperatures on the hatchability of shelf dried Artemia cysts from Kazakhstan
Introduction to Anaerobes
Anaerobic bacteria differ in their pathogenicity. Not all of them are believed to be clinically significant, while others are known to be highly pathogenic. Table 1 lists the major anaerobes that are most frequently encountered clinically. The taxonomy of anaerobic bacteria has changed in recent years because of their improved characterization using genetic studies (1). The ability to differentiate between similar strains enables better characterization of type of infection and predicted antimicrobial susceptibility. The species of anaerobes most frequently isolated from clinical infections are in decreasing frequency: the clinically important anaerobes are of gram-negative rods (Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutterella), gram-positive cocci (primarily Peptostreptococcus), gram-positive spore-forming (Clostridium) and non-spore-forming bacilli (Actinomyces, Propionibacterium, Eubacterium, Lactobacillus, and Bifidobacterium), and gram-negative cocci (mainly Veillonella) (2). About 95% of the anaerobes isolated from clinical infections are members of these genera. The remaining isolates belong to species not yet described, but these usually can be assigned to the appropriate genus on the basis of morphologic characteristics and fermentation products. The frequency of recovery of the different anaerobic strains differs in various infectious sites. The 12 years experience in recovering anaerobic bacteria from adults and children at two medical centers is presented in Table 2 (3). The main isolates were anaerobic gram-negative bacilli (Bacteroids, Prevotella, and Porphyromonas 43% of anaerobic isolates), anaerobic gram-positive cocci (26%), Clostridium spp. (7%), and Fusobacterium spp. (5%). This chapter discusses the main anaerobic species and their role in infectious processes.
CLASSIFICATION OF ANAEROBES
Anaerobes do not multiply in oxygen but have different susceptibility to oxygen. Most normal flora anaerobes are extremely oxygen sensitive, while those that cause infections are more aero-tolerant. The aero-tolerance of several anaerobes is through the production of superoxide dismutase, they produce on exposure to oxygen. The negative oxidation-reduction potential (Eh) of the environment is a critical factor in the survival of anaerobic bacteria.
Anaerobes do not grow on solid media in room air (10% CO2, 18% 02) facultative anaerobes grow both in the presence and absence of air, and microaerophilic bacteria grow poorly or not at all aerobically but grow better under 10% CO2 or anaerobically. Anaerobes are divided into "strict anaerobes" that are unable to grow in the presence of more than 0.5% 02 or "moderate anaerobes" that are capable of growing at between 2% and 8% 02.
GRAM-POSITIVE SPORE-FORMING BACILLI
Anaerobic spore-forming bacilli belong to the genus Clostridium. Morphologically, the clostridia are highly pleomorphic, ranging from short, thick bacilli to long filamentous forms, and are either ramrod straight or slightly curved. The clostridia found most frequently in clinical infections are Clostridium perfringens (Fig. 1), Clostridium septicum, Clostridium butyricum, Clostridium sordellii, Clostridium ramosum, and Clostridium innocuum.
C. perfringens is an inhabitant of soil and of intestinal contents of humans and animals and is the most frequently encountered histotoxic clostridial species (4). This microorganism, which
TABLE 1 Anaerobic Bacteria Most Frequently Encountered in Clinical Specimens Organism
Gram-positive cocci Peptostreptococcus spp. Microaerophilic streptococci3 Gram-positive bacilli Non-spore-forming Actinomyces spp.
Propionibacterium acnes Bifidobacterium spp. Spore-forming Clostridium spp. C. perfringens C. septicum C. sordellii C. difficile C. botulinum C. tetani Gram-negative bacilli Bacteroides fragilis group (B. fragilis,
B. thetaiotamicron) Pigmental Prevotella and Porphyromonas spp. Prevotella oralis Prevotella B. oris-buccae P. bivia, P. disiens Fusobacterium spp. F. nucleatum F. necrophorum
Respiratory tract, intra-abdominal and subcutaneous infections Sinusitis, brain abscesses
Intracranial abscesses, chronic mastoiditis, aspiration pneumonia, head and neck infections Shunt infections (cardiac, intracranial) Chronic otitis media, cervical lymphadenitis
Wounds and abscesses, sepsis Sepsis
Necrotizing infections Diarrheal disease, colitis Botulism Tetanus
Intra-abdominal and female genital tract infections, sepsis, neonatal infection Orofacial infections, aspiration pneumonia, periodontitis Orofacial infections
Orofacial infections, intra-abdominal infections Female genital tract infections
Orofacial and respiratory tract infections, brain abscesses, bacteremia Aspiration pneumonia, bacteremia
elaborates a number of necrotizing extracellular toxins, is easily isolated and identified in the clinical laboratory. C. perfringens seldom produces spores in vivo. It can be characterized in direct smears of a purulent exudate by the presence of stout gram-variable rods of varying length, frequently surrounded by a capsule. C. perfringens can cause a devastating illness with high mortality. Clostridial bacteremia is associated with extensive tissue necrosis, hemolytic anemia, and renal failure. The incidence of clostridial endometritis, a common event following septic abortions, has decreased as medically supervised abortions have increased (2).
C. perfringens accounted for 48% of all clostridial isolates in our hospitals (Table 2) and was primarily isolated from wounds (26% of C. perfringens) isolates, blood (16%), abdomen (14%), and obstetrical and gynecological infections (13%).
C. septicum, long known as an animal pathogen, has been found in humans within the last decade, often associated with malignancy. The intestinal tract is thought to be the source of the organism, and most of the isolates are recovered from the blood.
C. sordellii causes life threatening infections after trauma, childbirth, gynecological procedures, medically induced abortions, surgery and injection of elicit drugs. It can cause rapid progressive tissue necrosis, shock, multiorgan failure and death in about 3/4 of patients (4a).
Although Clostridium botulinum usually is associated with food poisoning, wound infections caused by this organism are being recognized with increasing frequency. Proteolytic strains of types A and B have been reported from wound infections. Disease caused by C. botulinum usually is an intoxication produced by ingestion of contaminated food (uncooked meat, poorly processed fish, improperly canned vegetables), containing a highly potent neurotoxin. Such food may not necessarily seem spoiled, nor may gas production be evident. The polypeptide neurotoxin is relatively heat labile, and food containing this toxin may be rendered innocuous by exposure to 100°C for 10 minutes.
TABLE 2 Percentage of Recovery of Anaerobes in Each Infection Site at Walter Reed Army Medical and Naval Medical Centers 1973-1985
Total number of anaerobic
Specimen number of anaerobic Isolates/ Bacteroides Fusobac- Clostridium Lactobacillus Eubacterium Propionibac- Bilidobac- Actinomyces Veillonella Peptostrepto-
source specimens Isolates specimen spp. /erfi/mspp. spp. spp. spp. /em/mspp. /em/mspp. spp. spp. coccj/sspp.
a In parentheses: percentage of all anaerobic bacteria isolated from source indicated. Source: From Ref. 3.
FIGURE 1 Gram stain of Clostridium per-fringens.
C. botulinum is usually associated with food poisoning (2) botulism is an intoxication caused by ingestion of contaminated food containing its highly potent neurotoxin. However, wound infections caused by proteolytic strains of types A and B has been reported with increasing frequency and can also produce botulism.
C. botulinum has also been associated with newborns presenting with hypotonia, respiratory arrest, areflexia, ptosis, and poorly responding pupils. Botulism in infants is caused by toxin from the germination of ingested spores and C. botulinum in the bowel lumen. C. butyricum can also be recovered from infection of the abdomen, abscesses, bile, wounds, and blood.
Clostridium difficile has been incriminated as the causative agent of antibiotic-associated and spontaneous diarrhea and colitis (5). A formerly infrequently isolated strain of C. difficile known as BI/NAP1 has recently been implicated in geographically diverse outbreaks of C. difficile-associated disease which have severe clinical presentations and poor outcomes (5).
Clostridium tetani is rarely isolated from human feces. Infections caused by this bacillus are a result of contamination of wounds with soil containing C. tetani spores. The spores will germinate in devitalized tissue and produce the neurotoxin that is responsible for the clinical findings of tetanus. C. tetani has been recovered from patients presenting with otogenous tetanus (6).
Clostridia can be isolated from various infectious sites. These organisms are especially prevalent in abscesses (mostly abdominal, rectal area, and oropharyngeal), and peritonitis (1). The distribution of clostridia in these infections is explained by their prevalence in the normal gastrointestinal and cervical flora from where they may originate (7).
Clostridia strains (C. perfringens, C. butyricum, and C. difficile) have been recovered from blood and peritoneal cultures of necrotizing enterocolitis and from infants with sudden death syndrome (8-10). Strains of Clostridium were recovered from children with bacteremia of gastrointestinal origin (11) and with sickle cell disease (12). Clostridial strains have been recovered from specimens obtained from patients with acute (13) and chronic (14) otitis media, chronic sinusitis and mastoiditis (15,16), peritonsillar abscesses (17), peritonitis (18,19), liver and spleen abscesses (20), abdominal abscesses (21), and neonatal conjunctivitis (22,23).
GRAM-POSITIVE NON-SPORE-FORMING BACILLI
Anaerobic, gram-positive, non-spore-forming rods comprise part of the microflora of the gingival crevices, the gastrointestinal tract, the vagina, and the skin. Since many of them appear to be morphologically similar, they have been difficult to separate by the usual bacteriologic tests. Several distinct genera are recognized: Actinomyces, Arachnid, Bifidobacterium, Eubacterium, Lactobacillus, and Propionibacterium.
The Actinomyces, Arachnia, and Bifidobacterium of the family Actinomycetaceae are grampositive, pleomorphic, anaerobic to microaerophilic bacilli. Species of the genus Bifidobacterium are part of the commensal flora of the mouth gastrointestinal tract and female genital tract and constitute a high proportion of the normal intestinal flora in humans, especially in breast-fed infants (24). Although some infections caused by these organisms have been reported (25-28), little is known about their pathogenic potential.
Eubacterium spp. are part of the flora of the mouth and the bowel. They have been recognized as pathogens in chronic periodontal disease (29) and in infections associated with intra-uterine devices (30), and have been isolated from patients with bacteraemia associated with malignancy (31) and from female genital tract infection (32). Lactobacillus spp. are ubiquitous inhabitants of the human oral cavity, the vagina, and the gastrointestinal tract (33). They have been implicated in various serious deep-seated infections, amnionitis (33) and bacteraemia (34). Eubacterium, Lactobacillus, and Bifidobacterium spp. have been isolated in pure culture in only a few instances and are usually isolated in mixed culture from clinical specimens (1). The infections where they have been found most often are chronic otitis media and sinusitis, aspiration pneumonia, and intra-abdominal, obstetric and gynecological and skin, and soft-tissue infections (1,35,36).
Actinomyces israelii and Actinomyces naeslundii are normal inhabitants of the human mouth and throat (particularly gingival crypts, dental calculus, and tonsillar crypts) and are the most frequently isolated pathogenic actinomycetes. These organisms have been recovered from intracranial abscesses (37), chronic mastoiditis (16), aspiration pneumonia (38), and peritonitis (18). Although actinomycetes often are present in mixed culture, they are clearly pathogenic in their own right and may produce widespread devastating disease anywhere in the body (39). The lesions of actinomycosis occur most commonly in the tissues of the face and neck, lungs, pleura, and ileocecal regions. Bone, pericardial, and anorectal lesions are less common, but virtually any tissue may be invaded a disseminated, bacteremic form has been described.
Propionibacterium spp. are part of the normal bacterial flora that colonize the skin (40), conjunctiva (41), oropharynx, and gastrointestinal tract (42). These non-spore-forming, anaerobic, gram-positive bacilli are frequent contaminants of specimens of blood and other sterile body fluids and have been generally considered to play little or no pathogenic role in humans.
Propionibacterium acnes and other Propionibacterium spp. have, however, been recovered with or without other aerobic or anaerobic organisms as etiologic agents of multiple infection sites (43-54). These include conjunctivitis (43), intracranial abscesses (44,45), peritonitis (46), and dental, parotid (47,48), pulmonary (47,48), and other serious infections (49). They have often been recovered as a sole isolate in specimens obtained from patients with infections associated with a foreign body (such as an artificial valve), endocarditis (50,51), and central nervous system shunt infections (50,52). The possible role of P. acnes in the pathogenesis of acne vulgaris was suggested. The data that support this are based on the recovery of this organism in large numbers from sebaceous follicles, especially in patients with acne, on its ability to elaborate enzymes such as lipase, protease, and hyaluronidase, and on its ability to activate the complement system and enhance chemotactic activity of neutrophils (53).
The anaerobic gram-negative rods are differentiated into genera on the basis of the fermentation acids they produce. The family Bacteroidaceae contains several genera of medical importance: Bacteroides fragilis group, Prevotella, Porphyromonas, Bacteroides, and Fusobacterium.
15.1.1: Gram Positive Rods - Biology
Chorioamnionitis due to Arcanobacterium haemolyticum
Sahira Haneefa, Resmi Rajan, Ramani Bai Joseph Theodore, Arya Raveendran Vasantha
Department of Microbiology, Govt. Medical College, Thiruvananthapuram, Kerala, India
|Date of Web Publication||3-Aug-2013|
Department of Microbiology, Govt. Medical College, Thiruvananthapuram, Kerala
Source of Support: None, Conflict of Interest: None
Chorioamnionitis can result either from the ascending of organisms from vagina after rupture of membrane or via the blood stream. This report describes a case of chorioamnionitis caused by Arcanobacterium haemolyticum, an unusual causative agent of chorioamnionitis. This is a case of a 22-year-old second gravida who was admitted for safe confinement at 34 weeks of gestation due to polyhydramnios. Passing of yellowish, foul smelling discharge intermittently was noticed. A. haemolyticum was isolated from amniotic fluid. Chorioamnionitis can result in significant maternal and fetal mortality and morbidity. Hence, it is important to ascertain the prompt diagnosis and treatment of suspected cases.
Keywords: Arcanobacterium haemolyticum , chorioamnionitis, premature rupture of membranes
|How to cite this article:|
Haneefa S, Rajan R, Theodore RB, Vasantha AR. Chorioamnionitis due to Arcanobacterium haemolyticum. J Acad Clin Microbiol 201315:34-5
|How to cite this URL:|
Haneefa S, Rajan R, Theodore RB, Vasantha AR. Chorioamnionitis due to Arcanobacterium haemolyticum. J Acad Clin Microbiol [serial online] 2013 [cited 2021 Jul 1]15:34-5. Available from: https://www.jacmjournal.org/text.asp?2013/15/1/34/116086
Chorioamnionitis is an infection of two membranes of the placenta (the chorion and the amnion) and the amniotic fluid that surrounds the baby. 
Arcanobacterium haemolyticum, an aerobic, slowly growing, catalase-negative Gram-positive bacillus, has been reported as an infrequent cause of peritonsillar abscess, pharyngitis, and tonsillitis in children and young adults.
Risk factors for the development of this infection remain to be identified. It is frequently a component of polymicrobial infection.  The organism, moreover, has been isolated from patients with chronic skin ulcers, soft tissue infections, deep tissue abscesses, meningitis, pneumonia, endocarditis, and bacteremia.
A 22-year-old second gravida at 34 weeks of gestation was admitted for safe confinement due to polyhydramnios. After 2 days of admission, the patient had labor pain and membrane ruptured following per vaginal examination. The discharge was yellowish and foul smelling. Fetal movements were noticed well by the patient. On examination, the patient was afebrile with uterine tenderness, heart rate was 100/min, and blood pressure was 120/80. Ultrasonography showed polyhydramnios. The hemoglobin and total leucocytes count were 9.0 and 10,000, respectively, at the time of admission. All other routine investigations (blood sugar, serum electrolytes, urea, and creatinine were within normal limits). The amniotic fluid, blood, and urine samples were collected and sent for culture and sensitivity. In the first pregnancy the antenatal period was uneventful, but the baby expired at 2 months due to sepsis.
Chorioamnionitis can result either from the ascending of organisms from vagina after rupture of membranes or via the blood stream. Commonly anerobes and group B streptococci have been reported as cause of chorioamnionitis.  Diagnosis of clinical chorioamnionitis is suggested by the presence of fever in a gravid patient without evidence of, or any other focus of infection. Ruptured membranes may or may not be present. Infective organism cannot be isolated from amniotic fluid in all cases.  The bacterial composition of amniotic fluid in case of ruptured membranes is often polymicrobial. But in this case only one type of organism was isolated.
A. hemolyticum is a Gram-positive rod having granular or beaded appearance. Colonies are beta hemolytic on blood agar and on Gram staining irregular club-shaped rod can be noticed.  Genus Arcanobacterium includes six species A. hemolyticum, A. pyogenes, A. bernardiae, A. phocae, A. pluranimalium, and A. hippocoleae. This organism was sensitive to Penicillin, Cephalosporins, Erythromycin, and Azithromycin. Macrolides have been proposed as the drug of choice, since treatment failure to beta lactum antibiotics have been reported. 
A bacterial staining procedure using crystal violet and pink safranin counterstain that generally divides bacteria into either gram-positive or gram-negative and useful for considering associated pharmacology. The procedure was named after Hans Christian Gram (1853 - 1938).
- Purple crystal violet stain is trapped by layer of peptidoglycan.
- peptidoglycan forms outer layer of the cell.
- Outer membrane prevents stain from reaching peptidoglycan layer in the periplasm.
- outer membrane is composed of four major components: lipopolysaccharide, phospholipids, beta-barrel proteins, and lipoproteins.
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