Aerobic and Anaerobic Microbiology of Surgical-Site Infection Following Spinal Fusion

The aerobic and anaerobic microbiology of surgical-site infections (SSI) following spinal fusion was retrospectively studied. This was done by reviewing the clinical and microbiological records at the Naval Hospital in Bethesda, Md., from 1980 to 1992. Aspirates of pus from 25 infection sites showed bacterial growth. Aerobic bacteria only were recovered from 9 (36%) specimens, anaerobic bacteria only were recovered from 4 (16%), and mixed aerobic and anaerobic bacteria were recovered from 12 (48%). Sixty isolates were recovered: 38 aerobes (1.5 isolates per specimen) and 22 anaerobes (0.9 isolate per specimen). The predominant aerobes were Escherichia coli (n = 8) and Proteus sp. (n = 7). The predominant anaerobes were Bacteroides fragilis group (n = 9) and Peptostreptococcus sp. (n = 6) isolates. An increase in recovery of E. coli and B. fragilis was noted in patients with bowel or bladder incontinence. This study highlights the polymicrobial nature of SSI and the importance of anaerobic bacteria in SSI following spinal fusion.     

How to isolate,identify and determine antimicrobial susceptibility of anaerobic bacteria in routine laboratories

BackgroundThere has been increased interest in the study of anaerobic bacteria that cause human infection during the past decade. Many new genera and species have been described using 16S rRNA gene sequencing of clinical isolates obtained from different infection sites with commercially available special culture media to support the growth of anaerobes. Several systems, such as anaerobic pouches, boxes, jars and chambers provide suitable anaerobic culture conditions to isolate even strict anaerobic bacteria successfully from clinical specimens. Beside the classical, time-consuming identification methods and automated biochemical tests, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has revolutionized identification of even unusual and slow-growing anaerobes directly from culture plates, providing the possibility of providing timely information about anaerobic infections.AimsThe aim of this review article is to present methods for routine laboratories, which carry out anaerobic diagnostics on different levels.SourcesRelevant data from the literature mostly published during the last 7 years are encompassed and discussed.ContentThe review involves topics on the anaerobes that are members of the commensal microbiota and their role causing infection, the key requirements for collection and transport of specimens, processing of specimens in the laboratory, incubation techniques, identification and antimicrobial susceptibility testing of anaerobic bacteria. Advantages, drawbacks and specific benefits of the methods are highlighted.ImplicationsThe present review aims to update and improve anaerobic microbiology in laboratories with optimal conditions as well as encourage its routine implementation in laboratories with restricted resources.     

Anaerobic infections in children: a prospective survey.

Over an 18-month period, cultures from 95 infants and children yielded 146 anaerobic organisms in 110 clinical specimens. Bacteroides was the most frequently isolated anaerobe, followed by Propionibacterium and Clostridium species. Intra-abdominal sources, soft tissues, and blood were the three major sources (82%) of isolation of anaerobes. Whereas most patients (58%) were over 5 years of age and only 11% were newborns, anaerobic infections constituted a rather uniform proportion of all infections, regardless of sources, in all age groups. Anaerobes accounted for only 2.9% of all positive cultures encountered from the various sources. Rates of recovery of anaerobes from intra-abdominal sources were significantly the highest, and from soft-tissue infections they were significantly the lowest. The anaerobic bacteremias observed were of no clinical significance when Propionibacterium species were isolated; however, recovery of other anaerobes from the blood, and primarily Bacteroides species, was usually associated with clinical disease. Except in blood cultures, anaerobes almost invariably coexisted with facultative bacteria.     

Isolation of Bacteroides ureolyticus (B corrodens) from clinical infections.

The introduction of an improved anaerobic system resulted in the isolation of Bacteroides ureolyticus (B corrodens) in numbers that suggested a pathogenic role from many more clinical specimens. During a three-year period B ureolyticus was isolated from 103 fairly superficial necrotic or gangrenous lesions all of which showed evidence of active infection. These included 27 perineal or genital infections, 15 perianal abscesses, 15 other soft tissue infections such as pilonidal abscesses and infected sebaceous cysts and 16 ulcers or gangrenous lesions of the lower limb. B ureolyticus was rarely isolated in pure culture but was usually one of the predominant organisms; the other organisms were mostly anaerobes and the combination of B ureolyticus with anaerobic Gram-positive cocci was particularly noticeable. The isolation and identification of B ureolyticus is not difficult but depends upon a reliable anaerobic system and the incubation of primary cultures for at least 72 h.     

Antimicrobial Resistance in Clinically Important Anaerobes

Antimicrobial resistance in anaerobes has increased during the past two decades; however, routine susceptibility testing is not performed in many clinical laboratories. Susceptibility testing of anaerobes is often limited to reference laboratories due to its labor-intensiveness and low testing volumes relative to susceptibility testing of other types of organisms, such as aerobic bacteria. Additionally, many polymicrobial anaerobic infections respond to debridement or drainage, and when antimicrobials are prescribed, knowing anaerobes are present in a specimen is often sufficient to drive treatment, a concept that has led to debate about the role of susceptibility testing for anaerobes. With the recent availability of commercial testing methods, more laboratories are performing anaerobic susceptibility testing. The majority of publications about resistance in anaerobes consist solely of in vitro surveys, and resistance can vary greatly between regions and even between hospitals in the same city. With increasing resistance in vitro, it is becoming more important to test isolates, monitor local susceptibility patterns, and perform clinical outcome studies.     

The bacteriology of infected malignant ulcers.

Infected ulcerated malignant tumours are often foul smelling and covered with necrotic tissue. We have studied 70 patients with infected ulcers; 30 of the underlying lesions in these patients were carcinoma of the breast, and 19 were a variety of skin cancers. Anaerobes were the predominant organisms isolated from individual ulcers. Of the 179 anaerobes isolated, 37 were Bacteroides asaccharolyticus, 31 each were B melaninogenicus and anaerobic streptococci, 29 B fragilis, and 17 B ureolyticus. Among the facultative organisms Escherichia coli was the commonest and was isolated mainly from patients with carcinoma of the breast. Most infections were mixed, yielding both anaerobes and aerobes and this made interpretation of the role of individual pathogens difficult to assess.     

Clinical significance ofStreptococcus milleri

The clinical features of infection in patients from whomStreptococcus milleri was isolated were analysed in an attempt to determine the clinical significance of this organism. During a four-year periodStreptococcus milleri was isolated from 232 hospitalized patients. In 44 patientsStreptococcus milleri was isolated in pure culture, in 45 patients together with obligate anaerobes, and in 143 patients together with aerobes with or without anaerobes. The 82 patients in whom isolation ofStreptococcus milleri was considered significant had the following infections: bacteremia (8 patients), brain abscess (2), pleural empyema (9), lung abscess (1), maxillary sinusitis (7), intra-abdominal abscess (53), infection of pacemaker (1) and infection of vascular graft (1). The 150 patients in whom isolation ofStreptococcus milleri was considered of questionable significance had the following: upper respiratory tract infection (12 patients), lower respiratory tract infection (6), acute cholecystitis (8), soft tissue abscess, cellulitis and surgical wound infections (83), bone and joint infections (14), genital infection (25) and miscellaneous infections (2). The overall incidence of infection withStrepococcus milleri was five cases per 1000 admissions. The study showed thatStreptococcus milleri is of clinical significance not only in suppurative infections, as previously reported, but also in acute maxillary sinusitis and infection of implant material.     

Mobiluncus species and other anaerobic bacteria in non-puerperal breast abscesses

Samples of 109 breast masses were cultured aerobically and anaerobically forMobiluncus spp. using selective and non-selective media. Two of the 103 evaluable samples were sterile and 57 (55%) yielded facultative bacteria only. The remaining 44 (43%) contained anaerobic growth alone or mixed infections. Seven (16%) of the 44 samples with anaerobes, six from women and one from a man, yieldedMobiluncus spp. Six were identified asMobiluncus curtisii and one asMobiluncus mulieris. There were two types of breast abscesses: one withStaphylococcus aureus predominating and one with anaerobes predominating. Most of the anaerobes belonged to the normal vaginal flora, but some were members of the normal oral flora. These results support the hypothesis that breast infections with these anaerobes result from sexual activities.     

Metronidazole-sensitive organisms in children with severe acute malnutrition: an evaluation of the indication for empiric metronidazole treatment

ObjectivesChildren with severe acute malnutrition (SAM) are treated with empiric amoxicillin or penicillin and gentamicin because of the high risk of severe infections. Experts have suggested, based on available evidence, adding metronidazole to cover anaerobic bacteraemia and diarrhoea caused by Giardia duodenalis or Clostridium difficile. The objective of this study was to assess the importance of these infections in children with SAM.MethodsChildren from 6 months to 15 years with SAM were enrolled and followed clinically. Aerobic and, when patient weight permitted, anaerobic blood cultures were done using Bactec® system, and isolates identified with matrix-assisted laser desorption ionization–time of flight mass spectrometry. Stool samples were tested for C. difficile, G. duodenalis and Entamoeba histolytica by PCR.ResultsA total of 334 children were enrolled and 174 out of 331 (53%) for which data on this was available had diarrhoea. Of 273 patients tested by blood culture, 11 had bacteraemia (4.0%, 95% CI 2.3–7.1%) but none with strict anaerobic bacteria (0/153, 95% CI 0–2.4%). There was no difference in the prevalence of C. difficile between children with (5/128, 4%) and without (7/87, 8%) diarrhoea (OR 0.47, 95% CI 0.14–1.53), and no difference in the prevalence of Giardia between these groups (78/138, 60% vs. 46/87, 53%; OR 1.34, 95% CI 0.77–2.32). Children with C. difficile had higher mortality than those without this infection (3/11, 27%, vs. 7/186, 4%; OR 43, 95% CI 3.9–483).ConclusionOur results do not provide support for empiric metronidazole to cover for anaerobic bacteraemia. Trials evaluating the effect of empiric treatment and its effect on G. duodenalis and C. difficile are warranted.     

In vitro activity of tigecycline against strains isolated from diabetic foot ulcers

Purpose of the studyThe aim of this study was to evaluate the in vitro activity of tigecycline and other comparator agents against bacterial strains isolated from diabetic foot infections (DFI).Patients and methodsAll diabetic patients hospitalized for a first episode of DFI (stage 2 to 4, according to the International Working Group of Diabetic Foot classification) were selected in Nîmes University hospital between January 2005 and June 2006. MICs were determined using custom broth microdilution panels against bacterial strains isolated from foot samples.ResultsThree hundred fifteen strains were studied. Tigecycline was active against 83.7% of all the strains especially Gram-positive cocci (97.3%) in particular methicillin-resistant Staphylococcus aureus (96%), Enterobacteriaceae (88.5%) and anaerobes (100%). Exclusively Pseudomonas aeruginosa and Proteae were not covered by this antibiotic.ConclusionsTigecycline, a new broad spectrum antimicrobial agent, is qualified to belong to the therapeutic arsenal package of complicated skin and soft tissue infections in diabetic patients after microbial documentation.     

Obligate anaerobes in clinical veterinary practice.

Clinical specimens obtained from domestic animals were examined to determine the relative prevalence of obligate anaerobic bacteria and the species represented. Of 3,167 samples cultured anaerobically as well as aerobically, 2,234 were bacteriologically positive. Of these positive samples, 583 (26%) contained species of obligate anaerobic bacteria in a total of 641 isolates. Most positive samples contained anaerobes admixed with aerobic species, although 6% of such samples yielded pure cultures of obligate anaerobes. The most common sites from which anaerobes were isolated were abscesses (32% of abscesses cultured contained species of obligate anaerobes), peritoneal exudates (24%), and pleural effusions (20%). Bacteroides melaninogenicus, Bacteroides spp., Peptostreptococcus anaerobius, and Bacteroides ruminicola accounted in the aggregate for approximately 50% of all anaerobic isolates. Bacteroides fragilis accounted for 1% of all the isolates, and members of the genus Clostridium accounted for 8%.     

Isolation and culture of Methanobrevibacter smithii by co-culture with hydrogen-producing bacteria on agar plates

ObjectivesMethanogenic Archaea are considered as extremely oxygen-sensitive organisms, and their culture is fastidious, requiring specific equipment. We report here conditions allowing the cultivation of Methanobrevibacter smithii in an anaerobic chamber without the addition of hydrogen.MethodsWe first enriched the stool sample in an anaerobic liquid medium. To cultivate M. smithii with Bacteroides thetaiotaomicron and other hydrogen-producing bacteria on solid medium in an anaerobic chamber, we divided the agar plates into two compartments and seeded each strain on each compartment. Methane production was assessed by gas chromatography, and the growing colonies were authenticated by MALDI-TOF MS.ResultsWe successfully cultured M. smithii from a liquid culture medium inoculated with stool collected from a healthy donor in an anaerobic chamber. The isolation in pure culture permitted successful culture on agar medium by our performing a co-culture with B. thetaiotaomicron. We also successfully tested the co-cultivation of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen in different amounts. Agar colonies of methanogens were obtained by co-culture with these bacteria, and methane production was detected.ConclusionsWe propose a new approach to isolate and cultivate new strains of M. smithii by using a co-culture–based technique that can facilitate and make available the isolation of new methanogenic Archaea strains in clinical microbiology laboratories.     

Bacteriological studies of gas gangrene and related infections

Gas gangrene, a life threatening condition is predominantly caused by Clostridium spp. Over a five year period (1996-2000), 580 wound swabs were processed from patients who had vehicular accidents with crush injury. The specimens were processed for anaerobic and aerobic organisms by standard laboratory techniques. Of the 580 swabs, 55(9.48%) were primary smear positive for spore bearing bacteria, of which 48 grew Clostridium spp. They were the sole anaerobic isolates in 30 patients (62.5%) whereas Clostridium spp. along with nonsporing anaerobes were isolated in 18 patients. Clostridium perfringens was the predominant isolate in 39 out of 48 cases (81.3%). However, 16 wound swabs which were primarily smear negative for spore bearers, grew Bacteriodes fragilis (11) and Fusobacterium species (5). All these 16 patients had foul smelling discharge with crepitation in the tissues. Our study highlights that apart from Clostridium spp., non-clostridial crepitant infections may present as myonecrosis simulating gas gangrene.     

Clinical significance of anaerobic bacteremias in a general hospital

Summary A prospective study was designed to investigate anaerobic bacteremias and evaluate their incidence and significance in a general hospital. One or more blood cultures positive for anaerobic microorganisms were analyzed from each of a total of 61 patients hospitalized between January 1988 and April 1992, in accordance with an established protocol. The clinical repercussions of bacteremia were also analyzed. Two percent of blood cultures were positive for anaerobes, with an incidence of 0.6 cases per 1000 hosphitalized patients. The most frequently isolated anaerobes were Bacteroides fragilis and Clostridium perfringens. Intraabdominal disease was the route of entry in 50% of the patients. A death rate of 37.3 % was mostly attributed to B. fragilis. Hospitalization in the surgical department, nosocomial acquisition, previous surgery, critical initial clinical status and the presence of complications were significantly associated with increased death rates. No significant differences were found in the clinical course between patients whose antibiotic treatment was judged adequate and those for whom it was considered inadequate. The frequency and incidence of anaerobic bacteremia was low in our hospital. The well-known clinical and epidemiological characteristics of these infections facilitates their prompt diagnosis and empirical treatment with antibiotics of proven effectiveness against anaerobes.     

Cardiac involvement in classical or hypermobile Ehlers–Danlos syndrome is uncommon

PurposeCardiac–valvular and vascular Ehlers–Danlos syndrome (EDS) have significant cardiovascular issues. The prevalence and significance of such abnormalities in classical (cEDS) or hypermobile EDS (hEDS) remain unclear. We report the prevalence of cardiac abnormalities in patients with cEDS and hEDS.MethodsWe identified 532 pediatric patients with potential EDS evaluated at our institution from January 2014 through April 2019 by retrospective chart review. Ninety-five patients (12 cEDS and 83 hEDS patients) met 2017 EDS diagnostic criteria and had an echocardiogram. One patient was excluded due to complex congenital heart disease, and two were excluded due to lack of images. We reviewed echocardiograms for all structural abnormalities.ResultsOf these 95 patients, 1 had mild aortic root dilation, and 1 had mild ascending aorta dilation in the setting of a bicuspid aortic valve. Eleven patients (11.6%) had a cardiac valve abnormality, all of which were trivial to mild. None of the patients required cardiac intervention.ConclusionOur results demonstrate that aortic dilation and valvular anomalies are uncommon in cEDS or hEDS patients. Given the lack of evidence, we do not recommend echocardiographic evaluation and surveillance in patients with cEDS and hEDS in the absence of clinical findings or positive family history.     

Anaerobes in pleuropulmonary infections

A total of 76 anaerobes and 122 aerobes were isolated from 100 patients with pleuropulmonary infections, e.g. empyema (64), pleural effusion (19) and lung abscess (13). In 14% of the patients, only anaerobes were recovered, while a mixture of aerobes and anaerobes was encountered in 58%. From all cases of lung abscess, anaerobic bacteria were isolated, alone (04) or along with aerobic bacteria (13). From empyema and pleural effusion cases, 65.6% and 68.4% anaerobes were recovered respectively. Amongst anaerobes, gram negative anaerobic bacilli predominated (Prevotella melaninogenicus 16, Fusobacterium spp. 10, Bacteroides spp. 9), followed by gram positive anaerobic cocci (Peptostreptococcus spp. 31). Coliform bacteria (45) and Pseudomonas aeruginosa (42) were the predominant aerobic isolates.     

Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system

Accurate and timely identification of anaerobic bacteria is critical to successful treatment. Classic phenotypic methods for identification require long turnaround times and can exhibit poor species level identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an identification method that can provide rapid identification of anaerobes. We present a multi-centre study assessing the clinical performance of the VITEK^® MS in the identification of anaerobic bacteria. Five different test sites analysed a collection of 651 unique anaerobic isolates comprising 11 different genera. Multiple species were included for several of the genera. Briefly, anaerobic isolates were applied directly to a well of a target plate. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry. Mass spectra results were generated with the VITEK^® MS, and the comparative spectral analysis and organism identification were determined using the VITEK^® MS database 2.0. Results were confirmed by 16S rRNA gene sequencing. Of the 651 isolates analysed, 91.2% (594/651) exhibited the correct species identification. An additional eight isolates were correctly identified to genus level, raising the rate of identification to 92.5%. Genus-level identification consisted of Actinomyces, Bacteroides and Prevotella species. Fusobacterium nucleatum, Actinomyces neuii and Bacteroides uniformis were notable for an increased percentage of no-identification results compared with the other anaerobes tested. VITEK^® MS identification of clinically relevant anaerobes is highly accurate and represents a dramatic improvement over other phenotypic methods in accuracy and turnaround time.     

CLAPO syndrome: identification of somatic activating PIK3CA mutations and delineation of the natural history and phenotype

PurposeCLAPO syndrome is a rare vascular disorder characterized by capillary malformation of the lower lip, lymphatic malformation predominant on the face and neck, asymmetry, and partial/generalized overgrowth. Here we tested the hypothesis that, although the genetic cause is not known, the tissue distribution of the clinical manifestations in CLAPO seems to follow a pattern of somatic mosaicism.MethodsWe clinically evaluated a cohort of 13 patients with CLAPO and screened 20 DNA blood/tissue samples from 9 patients using high-throughput, deep sequencing.ResultsWe identified five activating mutations in the PIK3CA gene in affected tissues from 6 of the 9 patients studied; one of the variants (NM_006218.2:c.248T>C; p.Phe83Ser) has not been previously described in developmental disorders.ConclusionWe describe for the first time the presence of somatic activating PIK3CA mutations in patients with CLAPO. We also report an update of the phenotype and natural history of the syndrome.     

Prospective evaluation of a novel multiplex real-time PCR assay for detection of fifteen respiratory pathogens—Duration of symptoms significantly affects detection rate

BackgroundNucleic acid amplification techniques have improved the diagnostic possibilities in respiratory tract infections, although their clinical applicability is not yet fully defined. We have evaluated a multiplex real-time PCR method for the detection of 13 respiratory viruses and 2 bacteria (Mycoplasma and Chlamydophila) in a clinical setting.ObjectivesThe aim of the present study was to evaluate the diagnostic performance and clinical use of a novel multiplex PCR method in adults with community-acquired respiratory viral infection, and the impact of duration of symptoms on detection rates.Study designNasopharyngeal swab samples were prospectively collected from 209 adult outpatients with respiratory infections and 100 asymptomatic controls.ResultsAn infectious agent was identified in 43% of samples from patients and 2% of asymptomatic controls. The detection rate was significantly higher in samples from patients with a duration of symptoms of 6 days or less (51%) than in samples from patients with a duration of symptoms of 7 days or more (30%, p < 0.01). For human corona viruses, and influenza virus A and B there was a correlation between the amount of virus in each patient sample as measured Ct values and duration of symptoms.ConclusionsDuration of symptoms significantly affects the detection rate of respiratory pathogens by multiplex real-time PCR in nasopharyngeal swab samples from adult patients with respiratory infections. Our finding should be taken into account when using these tests in clinical practise.     

microRNA expression profile in Smooth Muscle Cells isolated from thoracic aortic aneurysm samples

PurposeThoracic aortic aneurysm (TAA) is a cardiovascular disease characterized by increased aortic diameter, treated with surgery and endovascular therapy in order to avoid aortic dissection or rupture. The mechanism of TAA formation has not been thoroughly studied and many factors have been proposed to drive its progression; however strong focus is attributed to modification of smooth muscle cells (SMCs). Latest research indicates, that microRNAs (miRNAs) may play a significant role in TAA development – these are multifunctional molecules consisting of 19–24 nucleotides involved in regulation of the gene expression level related to many biological processes, i.e. cardiovascular disease pathophysiology, immunity or inflammation.Materials and methodsPrimary SMCs were isolated from aortic scraps of TAA patients and age- and sex-matched healthy controls. Purity of isolated SMCs was determined by flow cytometry using specific markers: α-SMA, CALP, MHC and VIM. Real-time polymerase chain reaction (RT-PCR) was conducted for miRNA analysis.ResultsWe established an isolation protocol and investigated the miRNA expression level in SMCs isolated from aneurysmal and non-aneurysmal aortic samples. We identified that let-7 g (0.71-fold, p = 0.01), miR-130a (0.40-fold, p = 0.04), and miR-221 (0.49-fold, p = 0.05) significantly differed between TAA patients and healthy controls.ConclusionsFurther studies are required to improve our understanding of the pathophysiology underlying TAA, which may aid the development of novel, targeted therapies. The pivotal role of miRNAs in the cardiovascular system provides a new perspective on the pathophysiology of thoracic aortic aneurysms.