Effect of prenatal cortisone on spontaneous bacterial translocation from gastrointestinal tract in neonatal rat

The immature host is prone to the passage of bacteria across the gut mucosal barrier. Corticosteroids accelerate the maturation of the intestinal mucosa and alter the composition of the gut bacterial flora. The present study was performed to assess the effect of prenatal cortisone on bacterial translocation in the neonatal rat. Time-pregnant Sprague Dawley rats were randomized on the 19th day of gestation for intraperitoneal injection of either 20 mg/100 g body weight of hydrocortisone or saline. Rats delivered spontaneously and the offspring were suckled ad libitum by the dam. Rat pups (N = 82) were killed 1 or 9 days after delivery. Mesenteric lymph nodes, liver, heart blood, and the terminal ileal loop were excised and quantitatively analyzed for bacteria. After one day, the proportion of rats with positive translocation was not significantly different between the two treatment groups (saline 62%, cortisone 80%, P = NS). By day 9, translocation had increased in the saline group (P = 0.03 vs day 1), did not significantly change in the cortisone group, and was significantly lower in rats treated with cortisone compared with the saline control (saline 90%, cortisone 60%, P = 0.02). The decrease in bacterial translocation after treatment with cortisone was associated with significantly lower total bacterial counts in the ileum (P < 0.05). Cortisone did not reduce bacterial counts in extraintestinal organs with positive translocation. In conclusion, prenatal treatment with cortisone reduces the incidence of spontaneous bacterial translocation from the intestine but not the concentration of translocated bacteria in extraintestinal organs of 9-day-old rats. Cortisone-induced changes of the intestinal microflora may have contributed to the reduction in translocation frequency.     

Changes in the bacterial flora of the throat and intestinal tract during prolonged oral administration of penicillin

A bacteriologic study of the throat and intestinal flora of ten patients with arthritis, treated orally with 500,000 to 1,000,000 units of penicillin daily over a prolonged period of time, has been carried out.Upon administration of penicillin there was a sudden change in the bacterial flora of the throat from gram-positive to gram-negative. Coliform bacteria appeared in the throat early in the course of treatment and were present, and often predominant, throughout the course. Subsequent to treatment there was a rapid reappearance of gram-positive cocci in the throat. The coliform organisms disappeared gradually.There was a lower incidence of streptococcus-positive stool cultures during, than prior to or subsequent to penicillin therapy.No difference in the range of penicillin sensitivity of the gram-positive cocci isolated from throat cultures prior to and subsequent to treatment was demonstrated. Penicillin-sensitive organisms were infrequent in the throat and stool cultures during treatment.There was no evidence that bacterial resistance to penicillin developed during the course of therapy.     

Role of intestinal bacterial overgrowth and intestinal motility in bacterial translocation in experimental cirrhosis.

BACKGROUND: Intestinal bacterial overgrowth (IBO) is related to small bowel motility and has been involved in the pathogenesis of bacterial translocation (BT) in experimental models, and both overgrowing gut flora and translocating bacteria to mesenteric lymph nodes are common features in cirrhosis. OBJECTIVES: The aims of this study were to analyze cecal aerobic bacteria and intestinal transit in cirrhotic rats, and their relationship with BT, evaluating the role of intestinal bacterial overgrowth and small bowel dismotility in the development of BT in experimental cirrhosis. MATERIAL AND METHODS: We included twenty-seven male Sprague-Dawley rats with carbon tetrachloride-induced cirrhosis without ascites and ten controls. Cultures of mesenteric lymph nodes (MLN), peripheral and portal blood, liver, spleen and cecal samples were carried out. Small intestinal transit was determined in ten cirrhotic rats and in ten control rats. RESULTS: The prevalence of bacterial translocation was 56%. Total cecal aerobic bacteria count was significantly higher in cirrhotic rats than in control rats (p < 0.001). Cirrhotic rats with translocated bacteria had higher total aerobic intestinal counts than culture-negative MLN bacteria (p < 0.05). The prevalence of total intestinal bacterial overgrowth in cirrhotic animals was 67%, and 0% in control animals (p < 0.001). According to BT, total IBO was more frequent in cirrhotic rats with BT versus those without BT (93 vs. 33%) (p < 0.001). Of the translocating bacteria, 95.6% were found to be overgrown in the cecum. The small-intestinal transit was slower in cirrhotic rats (60.5 +/- 12.7 cm vs. 81.2 +/- 5.7 cm) than in control animals (p < 0.001). CONCLUSIONS: These results suggest that the increase of intestinal aerobic bacteria in experimental cirrhosis is associated with translocation. In addition, IBO is frequent in cirrhotic rats, and is supposed to play an important role in the development of BT. Impaired motility of the small intestine is a common feature in cirrhosis and may be implicated in the pathogenesis of IBO.     

Effect of Lactobacillus-fermented diets on bacterial translocation and intestinal flora in experimental prehepatic portal hypertension

Spontaneous bacterial infections in cirrhosis and portal hypertension have been attributed to trans- location of gut-derived bacteria, a process promoted by intestinal bacterial overgrowth and disruption of the gut mucosal barrier. Bacteriotherapy with Lactobacillus has been reported to correct bacterial overgrowth, stabilize mucosal barrier function, and decrease bacterial translocation in rat models of acute liver injury and failure. In this study we investigated the effect of Lactobacillus-supplemented diets on intestinal flora and on bacterial translocation rate in portal vein ligated rats. Lactobacillus-fermented milk (yogurt) containing at least 2 × 10⁹ colony forming units/ml or placebo (water) was adminstrated by gavage twice daily (2 ml) for 9 days. Portal vein ligation was performed on day 7 of treatment. Bacterial translocation to mesenteric lymph nodes and quantification of intestinal flora was assessed by standard bacteriological cultures. Bacterial translocation was not significantly different between animals that received yogurt (82%) and those that received placebo (75%). Yogurt did not induce any significant changes in intestinal flora, whether it was produced with Lactobacillus acidophilus or Lactobacillus GG. In conclusion, in acute prehepatic portal hypertension, bacteriotherapy with Lactobacillus was unable to induce changes in bacterial translocation probably because it was unable to induce changes in bacterial flora.     

Intestinal bacterial overgrowth and bacterial translocation in cirrhotic rats with ascites

Background/Aims: Translocation of indigenous bacterial from the gut lumen of cirrhotic animals to mesenteric lymph nodes appears to be an important step in the pathogenesis of spontaneous bacterial peritonitis. However, the sequence of events leading to translocation remains unclear. One of the most predictable risk factors for translocation is overgrowth of gut bacterial flora. The present study was designed to compare the intestinal aerobic bacterial flora of cecal stools at the time of sacrifice between cirrhotic and normal rats and to evaluate the role of intestinal aerobic bacterial overgrowth in bacterial translocation in cirrhotic rats.Methods: Thirty-five male Sprague-Dawley rats with carbon tetrachloride-induced cirrhosis and ascites and 10 normal rats were included in this study. Cirrhotic rats were sacrificed when ill and samples of ascitic fluid, mesenteric lymph nodes and cecal stool were taken for detecting quantitatively aerobic bacteria.Results: Total intestinal aerobic bacterial count in cecal stool at the time of sacrifice was significantly increased in cirrhotic rats with bacterial translocation with or without spontaneous bacterial peritonitis compared to cirrhotic rats without bacterial translocation (p<0.001 and p<0.001, respectively) and to normal rats (p<0.001 and p<0.001, respectively). Of the 42 species of bacteria translocating to the mesenteric lymph nodes, 41 (97.6%) were found in supranormal numbers in the stool at the time of sacrifice.Conclusions: Carbon tetrachloride-induced cirrhotic rats with bacterial translocation have increased total intestinal aerobic bacteria count, and intestinal bacterial overgrowth appears to play an important role in bacterial translocation in this experimental model of cirrhosis in rats.     

Ciprofloxacin for selective decontamination of the alimentary tract in patients with acute leukemia during remission induction treatment: the effect on fecal flora

Ciprofloxacin, a new quinolone derivative, was given prophylactically (500 mg twice a day) to 15 patients with acute leukemia during remission induction treatment for a mean duration of 42 days. The effect on the microbial flora of the alimentary tract was evaluated. A rapid elimination of Enterobacteriaceae within three to five days was observed. Bacteroides and Clostridium species were not affected, but the number of anerobic nonsporeforming gram-positive bacilli and anaerobic cocci was decreased. Nine ciprofloxacin-resistant gram-negative rods (Pseudomonas and Acinetobacter species) were isolated but without colonization or subsequent infection. Four of the five bacteriologically documented infections in these patients were caused by gram-positive cocci. Ciprofloxacin was well tolerated, and no side effects were noticed.     

Effect of selective bowel decontamination with norfloxacin on spontaneous bacterial peritonitis,translocation, and survival in an animal model of cirrhosis

Selective bowel decontamination with the orally administered quinolone antibiotic, norfloxacin, has been shown to suppress gut gram-negative bacteria and help prevent gram-negative infections in cirrhotic patients who are at high risk of bacterial infection. Because this drug does not eradicate gram-positive organisms, it is conceivable that gram-positives could replace the suppressed gram-negatives in the gut and lead to subsequent infection. Also the effect of norfloxacin on translocation (as defined by culture positivity of mesenteric lymph nodes) has received little attention. In this study, the effect of oral norfloxacin on translocation, bacterial peritonitis, and survival was investigated in an animal model of carbon tetrachloride—induced cirrhosis and ascites. Treated rats received daily doses of orally administered norfloxacin from the onset of cirrhosis until they died or were killed. Controls received no antibiotic. Norfloxacin led to a reduction in bacterial peritonitis from 70% in untreated cirrhotic controls to 28% in treated cirrhotic rats; these data were statistically significant (P = .012). There was no effect on overall translocation rate (28% with norfloxacin vs. 50% without norfloxacin) (P > .1). Gram-positives were isolated in 100% of the bacterial peritonitis episodes and in 71.4% of culture-positive mesenteric lymph nodes in treated animals compared with only 25% of peritonitis episodes and 10% of culture-positive mesenteric lymph nodes of untreated cirrhotic controls (P < .01 for peritonitis and P < .05 for translocation). The survival rate was not different between groups (P > .1). Although norfloxacin was associated with a 60% reduction in the overall rate of peritonitis, this drug increased the risk of gram-positive translocation and gram-positive peritonitis, and did not prolong survival in this animal model.     

Leaky gut and the liver: a role for bacterial translocation in nonalcoholic steatohepatitis

Gut flora and bacterial translocation (BT) play important roles in the pathogenesis of chronic liver disease, including cirrhosis and its complications. Intestinal bacterial overgrowth and increased bacterial translocation of gut flora from the intestinal lumen predispose patients to bacterial infections, major complications and also play a role in the pathogenesis of chronic liver disorders. Levels of bacterial lipopolysaccharide, a component of gram-negative bacteria, are increased in the portal and/or systemic circulation in several types of chronic liver disease. Impaired gut epithelial integrity due to alterations in tight junction proteins may be the pathological mechanism underlying bacterial translocation. Preclinical and clinical studies over the last decade have suggested a role for BT in the pathogenesis of nonalcoholic steatohepatitis (NASH). Bacterial overgrowth, immune dysfunction, alteration of the luminal factors, and altered intestinal permeability are all involved in the pathogenesis of NASH and its complications. A better understanding of the cell-specific recognition and intracellular signaling events involved in sensing gut-derived microbes will help in the development of means to achieve an optimal balance in the gut-liver axis and ameliorate liver diseases. These may suggest new targets for potential therapeutic interventions for the treatment of NASH. Here, we review some of the mechanisms connecting BT and NASH and potential therapeutic developments.     

Gut flora and bacterial translocation in chronic liver disease

Increasing evidence suggests that derangement of gut flora is of substantial clinical relevance to patients with cirrhosis. Intestinal bacterial overgrowth and increased bacterial translocation of gut flora from the intestinal lumen, in particular, predispose to an increased potential for bacterial infection in this group. Recent studies suggest that, in addition to their role in the pathogenesis of overt infective episodes and the clinical consequences of sepsis, gut flora contributes to the pro-inflammatory state of cirrhosis even in the absence of overt infection. Furthermore, manipulation of gut flora to augment the intestinal content of lactic acid-type bacteria at the expense of other gut flora species with more pathogenic potential may favourably influence liver function in cirrhotic patients. Here we review current concepts of the various inter-relationships between gut flora, bacterial translocation, bacterial infection, pro-inflammatory cytokine production and liver function in this group.     

The effect of minimum luminal nutrition on bacterial translocation and atrophy of the jejunum during parenteral nutrition

In situations of catabolic stress, the gut becomes atrophic and may have diminished barrier function as evidenced by an increase in bacterial translocation. The aim of this study was to examine the effect of minimum luminal nutrition during parenteral nutrition on the extent of jejunal atrophy and rate of bacterial translocation. Central venous lines were inserted into 30 rats before they underwent randomization to receive nutritional support with: (a) conventional parenteral nutrition; (b) conventional parenteral nutrition with 3 g/day of rat food (i.e., minimum luminal nutrition); or (c) rat food ad libitum. The rats were assessed after 10 days for nutritional status, extent of jejunal atrophy, caecal flora, as well as the extent of bacterial translocation to the mesenteric lymph nodes, liver and spleen. Rats in the rat food ad libitum group lost the smallest amount of weight and had the least amount of jejunal atrophy, yet had a similar rate of bacterial translocation as the parenterally nourished groups. When compared with the conventional parenteral nutrition group, the minimum luminal nutrition group had better preservation of the weight of the small bowel and its isolated mucosa (P < 0.01), but had a similar rate of bacterial translocation. Minimum luminal nutrition reduced the extent of atrophy of the gut but did not affect the incidence of bacterial translocation. It is inferred that there is no direct relationship between the extent of mucosal atrophy and incidence of bacterial translocation.     

Mucosal colonisation with Lactobacillus casei mitigates barrier injury induced by exposure to trinitronbenzene sulphonic acid

BACKGROUND: Trinitrobenzene sulphonic acid (TNBS) induces chronic transmural inflammatory lesions in the rat colon. Injury is facilitated by barrier disruption and invasion of commensal bacteria. However, certain bacteria have shown anti-inflammatory properties in in vitro models. AIM: To investigate in vivo the anti-inflammatory effect of Lactobacillus casei DN-114 001. METHODS: Rats with a colonic segment excluded from faecal transit were surgically prepared. After washing the lumen with antibiotics, the excluded segment was recolonized (control group: standard flora of rat origin; test group: standard flora and L casei). Microbial colonisation was confirmed by culture of segment washing, and colitis was then induced by instillation of TNBS. One day after, intestinal lesions were blindly graded by macro- and microscopic scores, and myeloperoxidase activity measured in tissue homogenates. Translocation of bacteria to mesenteric lymph nodes, spleen and liver was investigated. RESULTS: Test rats showed a smaller area of mucosal injury than control rats (p<0.05). Maximum depth lesion scores were similar in both groups but myeloperoxidase activity was lower in test than in control rats (p<0.05). Remarkably, bacterial translocation was quantitatively lower (p<0.01) and less frequent (p<0.05) in test than in control rats. CONCLUSION: In rats colonised with L casei, mucosal injury, inflammatory response, and barrier disruption after TNBS challenge were attenuated. Bacterial communities colonising the mucosa can modify inflammatory responses to luminal challenges.     

Alterations in rat intestinal transit by morphine promote bacterial translocation

Translocation of enteric microorganisms from the intestinal tract to extraintestinal sites has been proposed as an early step in the development of gram-negative sepsis. This study examined the role of altered bowel transit in influencing intestinal bacteriostasis and bacterial translocation using morphine as a pharmacologic inhibitor of such transit. In the first experiment, either normal saline (N=8) or morphine sulfate (20 mg/kg;N=8) was injected subcutaneously. Two hours later, morphine (7.5 mg/kg) was infused subcutaneously for an additional 22 hr; control animals received saline alone. After completion of this regimen, a volume of 0.2 ml of 2.5 mM FITC dextrans (10,000 daltons) were injected intraduodenally in each group. The bowel was removed 25 min later, divided into 5-cm segments, and the content of dextrans measured. Small bowel propulsion was expressed as the geometric center of the distribution of dextrans throughout the intestine (in percentage length of small bowel). Gut propulsion was significantly reduced after morphine treatment as compared to controls (32.8±8.2% vs 55.8±4.0%;P<0.01). In 16 additional rats, saline or morphine was again administered as described. After 24 hr, samples were obtained from the mesenteric lymph node (MLN) complex, blood, spleen, liver, duodenum, jejunum, ileum, and cecum for standard bacteriology. The bacterial counts increased significantly in each intestinal segment following morphine treatment. Microorganisms translocated to the MLN complex in 5, and to distant sites in four of eight morphine-treated animals, respectively. Translocation to the MLN complex occurred in only one of eight controls (P<0.05); no translocation to distant sites occurred in control animals. We conclude that the morphine-induced prolongation in bowel transit promotes bacterial translocation secondary to an overgrowth of enteric bacteria in the intestinal lumen.Supported by NIH grant GM 38529 and the DFG grant Ru 387/1-2.     

Effect of Chinese herbal mixture, shock decoction on bacterial translocationfrom the gut

AIM In order to provide the TCM therapeutic basis for MODS in clinical critical patients, the role of shockdecoction in anti-bacterial translocation from the gut was tested in rats.METHODS Based on the pathophysiology of MODS following bacterial translocation from the gut causedby severe injuries such as burn, shock, hemorrhagic shock model that induced obvious bacterial translocationwas established and used to determine whether shock decoction, that is composed of modified WenpiDecoction, reduces bacterial translocation. Bacterial culture for mesenteric lymph nodes, liver and spleen ofrats in shock, treatment and control groups was used to calculate the incidence of bacterial translocation.RESULTS The incidence of intestinal bacteria translocating to mesenteric lymph nodes, liver and spleenwas lower in the shocked rats infused via gastrogavage with shock decoction (3/ 15) than that in thenoninfused shocked rats (11 / 13), (P = 0.0009, < 0.01 ). The incidence of intestinal bacteria translocationof rats in shock and control groups were distinctly different (P = 0.0017, <0.01). The amounts and speciesof intestinal flora between infused and noninfused shocked rats were not different statistically (P=0.101,P>0.05). Histological examination showed that intestinal mucosa edema was severer in the shocked ratsthan in the shocked rats with gastrogavage.CONCLUSION Shock beverage could inhibit the shock-induced enterogenous bacterial translocation in ratsprobably by its protective role in intestinal mucosa structure; and has no effect on the growth of intestinalbacteria.     

Modification of small bowel microflora in chronic alcoholics with alcoholic liver disease.

The bacterial flora of the gastrointestinal tract plays an essential role in human physiology. Our aim was to study the pattern of change in bacterial microflora in the small intestines of patients with chronic alcoholic liver disease (ALD). Patients of ALD made up the test group and nonalcoholics served as controls. Duodenal (D2) biopsies were obtained by upper gastrointestinal endoscopy and processed immediately for microbiological analysis. Marked qualitative and quantitative alterations of small intestinal microflora was documented in chronic alcoholics. There was increased bacterial growth of both gram-positive cocci and gram-negative bacilli in the ALD group.     

Bacterial carriage by artificial versus natural nails

Although health care workers are following fashion trends when they wear artificial nails, the implications of these nails for infection control is unknown. Cultures were obtained before and after handwashing from the fingertips of 56 nurses with artificial nails and 56 nurses with natural nails. After handwashing there were higher numbers of colony-forming units of gram-negative rods cultured from the fingertips of nurses with artificial nails than from nurses with natural nails. There were no significant differences in carriage of gram-positive cocci or normal flora. Because of the number of nosocomial infections caused by gram-negative rods, health care workers who wear artificial nails should consider the potential risk of increased carriage of gram-negative rods.     

Gram-positive cocci are associated with the formation of completely pure cholesterol stones

OBJECTIVES: Recently it has been reported that bacterial DNA has been detected in mixed cholesterol stones (cholesterol content < 95%), which were not previously believed to be related to bacteria, using the polymerase chain reaction (PCR). We examined bacterial DNA in pure cholesterol stones to clarify the mechanism of initiation or promotion of the formation of cholesterol gallstones. METHODS: We examined 69 gallstones (30 brown pigment stones, 21 pure cholesterol stones, and 18 mixed cholesterol stones). Bacterial DNA was extracted from the core of the gallstones and amplified by PCR. Bacteria species in gallstones were identified by DNA sequencing of the PCR products. RESULTS: Bacterial DNA was detected in 26/30 brown pigment stones (87%), in 12/21 pure cholesterol stones (57%) (cholesterol content = 100%), and in 12/18 mixed cholesterol stones (67%) (cholesterol content = 82-95%). Bacterial species in gallstones were identified by DNA sequencing of PCR products. Eighty percent of bacteria in brown pigment stones were gram-negative rods or anaerobes. In contrast, 100% of bacteria in pure cholesterol stones were gram-positive cocci. The bacteria in mixed cholesterol stones consisted of 40% gram-positive cocci, 50% gram-negative rods, and 10% anaerobes. CONCLUSIONS: It was strongly suggested that gram-positive cocci are associated with the formation of pure cholesterol stones.     

Canine granulopoiesis: alterations induced by suppression of gram-negative flora.

We investigated alterations in canine granulopoiesis following suppression of bacterial flora by antibiotic treatment. Beagles were decontaminated in sterile isolation with laminar air flow, by an antifungal and antibiotic regimen. Skin and fecal specimen cultures were usually negative after 4 days. The reduction of gram-negative bacteria resulted in a significant decrease in plasma colony-stimulating factor (CSF) levels, marrow CFU-c concentration, and the cytopoietic activity of marrow-derived diffusion chamber (DC) progenitor cells. The period of conventionalization was characterized by a significant although delayed increase in plasma CSF as well as marked increases in marrow CFU-c concentration and cytopoietic activity of marrow DC progenitors. There was also mobilization of marrow DC progenitors into the circulation. All parameters returned to control values within 7 days of conventionalization. These data supported the hypothesis that the gram-negative bacteria of the gut play a significant role in the regulation of normal canine granulopoiesis.     

重症监护病房院内感染调查分析

目的 :研究重症监护病房院内感染菌群的变迁及细菌耐药的情况 ,为经验性选择抗生素提供依据 ,以达到控制院内感染的目的。方法 :对本院ICU 13 9例患者分离出的 3 10株细菌进行分析。结果 :共分离出革兰阳性 (G+)球菌 64株 ,占总数的 2 0 .6% ;革兰阴性 (G-)杆菌共 178株 ,占总数的 5 7.5 %。G-杆菌中列前三位的分别是铜绿假单胞菌 ( 2 1.6% ) ,肠杆菌 ( 6.8% ) ,大肠艾希菌( 6.5 % )。真菌共 61株 ,占总数的 19.7%。亚胺培南 西拉司丁钠、阿米卡星、头孢他啶对所有的G-杆菌保持着良好的抗菌活性 ,而环丙沙星的敏感率逐年下降。院内感染的发生部位以呼吸道最为常见。结论 :感染的细菌仍以G-杆菌为主。真菌的感染率始终较高 ,并且出现菌种的变异。菌种及细菌药敏的变化使经验性的选择抗生素变得越来越困难。临床医师应尽量减少用药的盲目性 ,以控制耐药菌的产生     

The effects of chronic bacteremia on the metabolic response to exercise in the conscious rat

Cardiac function was indirectly assessed in a rat model of chronic sepsis by measuring maximal oxygen uptake (VO2max) during exercise. A subcutaneous abscess cavity was created in rats by implanting a gauze sponge in the hindquarter area. Animals with sterile abscesses were compared with rats that had the abscess cavity infected with Escherichia coli and Bacteroides fragilis (gram-negative group) or these two organisms plus Staphylococcus aureus (gram-positive/negative group). VO2max was measured during exercise before and after the abscess cavity was infected. After a carotid artery cannula was placed, heart rate, mean arterial pressure, arterial blood gases, and acid-base status were measured in three groups of rats (sterile abscess, gram-negative, gram-positive/negative) during submaximal and maximal exercise. Results demonstrated that the infected rats were febrile and had elevated white blood cell counts and intermittent bacteremia, while rats with sterile abscesses did not. VO2max was similar in all groups of rats both before and after the abscess cavity was infected. The metabolic and hemodynamic variables measured during submaximal and maximal exercise in the different groups of rats were similar. These results do not support the contention that gram-positive and/or-negative bacteremia produce cardiac dysfunction during early sepsis.     

The clinical importance of anaerobic bacteria in wound infections after gastrointestinal surgery.

Wound cultures from 54 patients with infections after gastrointestinal surgery were examined. Cultures from wounds after surgery on the upper gastrointestinal tract grew few organisms, mainly aerobic gram-positive cocci. Culture from wounds on the lower gastrointestinal tract grew strains of bacteria, aerobic and anaerobic gram-negative rods dominating. Indirect immunofluorescence studies on acute and convalescent phase sera showed significant immune response against Bacteroides fragilis in a majority of cases. Immune response against anaerobic cocci was seldom found. Very high antibody titres against Clostridium perfringens were often found, both in patients' and control sera.