Evaluation of Amniotic Fluid for Aerobic and Anaerobic Bacteria JAY





B.S., MT. (ASCP),

AND P H Y L L I S M I L L E R , M.D. From the Departments of Pathology and Obstetrics and Gynecology, Baroness Erlanger Hospital, Chattanooga, Tennessee


FLUID from the intact amniotic cavity is ordinarily considered sterile. 13 In previous studies t h e methods for isolation a n d identification of anaerobic bacteria in amniotic fluid were either inadequate by current standards, or were incompletely described in the article. This study was u n d e r t a k e n to clarify t h e state of anaerobic flora in amniotic fluid, and to evaluate the usefulness of amniocentesis as an early indication of infection, due to anaerobic or facultative anaerobic or aerobic organisms, and the relationship to neonatal infection. Received April 11, 1975; accepted for publication

April 24, 1975.

Methods and Materials A f

e w private patients were evaluated, e majority of the patients were from th e clinic service at the Baroness Erlanger Hospital. The patients were neither randomized nor selected. The majority of the patients had amniocentesis for evaluation of fetal maturity. A few had amniocentesis at the time of a second-trimester abortion, Others had amniocentesis for evaluation of bacterial flora following rupture of the amniotic membranes. Some were done at the time of cesarean section; these cultures were obtained after the skin and

T h




incised b u t

prior to entering the uterus. The technics

Presented in part at the 74th Annual Meeting of ... . r the American Society for Microbiology, Chicago, U t i l i z e d , e x c e p t for p a t i e n t s

. undergoing

Illinois, May 12-17, 1974.

cesarean section, were: (1) The patient

Address reprint requests to Dr. Lewis: Department of Pathology, Baroness Erlanger Hospital,

w a s

Chattanooga, Tennessee 37403.

The baby was located by manual examina58

• t ,. j . . . . . U L I J J /O\ instructed to empty the bladder. (2)

Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016

Lewis, Jay F., Johnson, Peggy, and Miller, Phyllis: Evaluation of amniotic fluid for aerobic and anaerobic bacteria. Am J Clin Pathol 65: 5 8 - 6 3 , 1976. Studies of 117 pregnant women, 83 at term, were instituted to determine the bacteriologic state of amniotic fluid, utilizing both standard aerobic and anaerobic technics. A high association of postpartum infection was found in women who had long periods of premature ruptured membranes and many vaginal examinations. Significant organisms including anaerobes, were isolated in many of these instances. Based on the findings of these studies, it is recommended that amniocentesis for aerobic and anaerobic cultures be done when membranes have been ruptured for 8 hours or more, and when the patient has had seven or more vaginal examinations during the course of labor. (Key words: Amniotic fluid; Postpartum infection; Anaerobic infection; Amnionitis.)

January 1976



All material received in the anaerobic laboratory was handled in the same manner. Anaerobic technics used were based on the Virginia Polytechnic Institute methods. 6 Amniotic fluid was inoculated in a prereduced anaerobically sterilized roll tube.$ In addition, the material was inoculated into PRAS chopped-meat glucose, t Both of these inoculations were made under constant flow of oxygen-free carbon dioxide using a Belco§ apparatus. In addition, the material was inoculated onto prereduced blood agar plates containing palladium catalyst^ and incubated in BBLH anaerobic jars. The fluid was also placed into trypticase soy broth** and blood agar plates for aerobic incubation. The aerobic and facultative anaerobic organisms were identified using standard schemes. 1 The anaerobes were identified using standard biochemical procedures, and gas chromatography procedures as per the Virginia Polytechnic Institute Anaerobe Manual. 8 Gram stains were examined on all fluids. * Gould Inc., Palo Alto, California. t The Purdue Frederick Co., Norwalk, Connecticut. t Scott Laboratories, Fiskville, Rhode Island. § Bellco Glass Co., Inc., Vineland, New Jersey. H Baltimore Biological Laboratories, Cockeysville, Maryland. ** Difco Corporation, Detroit, Michigan.

Table 1. Patients with Postpartum Infection with Positive Culture Time Ruptured P.T.A*

Vaginal Exams P.T.A.*

Bacteroides sp. Enterococcus

28 hours



P. intermedhis

15 hours



Bacteroides sp. Enterococcus

13 hours



Lactobacillus sp.

8 hours



Klebsiella sp. E. coli P. mirabilis Enterococcus

1 1 hours



E. coli

Case 6

Organism Isolated

24 hours



S. epidermidis

o hours



E. coli

6 hours


* P.T.A. = prior to amniocentesis.

Results A total of 117 patients was evaluated. Eighty-three of these were at term or near term. Eight of the term patients (9.7%) had courses consistent with postpartum infection. Pathogens were demonstrated in the samples of amniotic fluid from six of the eight. In one case of postpartum infection that could not be otherwise explained, Lactobacillus sp. was isolated on two occasions. The organism could have been introduced at the time of the insertion of the internal catheter. However, Sharpe and associates" have demonstrated that Lactobacillus can be a pathogen under certain conditions. In one case, Staphylococcus epidermidis was isolated. We assume that it was a contaminant. In all cases of infection, the amniotic sac had been ruptured for prolonged periods prior to amniocentesis—the shortest period was 8'/2 hours. In each instance of infection, the patient had had 7 or more vaginal examinations prior to amniocentesis (Table 1). Patients with clinical and bacteriologic evidence of infection had an

Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016

tion. (3) The placenta was located by utilizing a doptone.* (4) The abdomen was prepared with Betadinef solution, which was allowed to dry. (5) The patient was d r a p e d with sterile drapes. (6) Lidocaine, 1%, was used for local anesthesia. A 22-gauge spinal needle with a stylet was inserted into the amniotic cavity. The stylet was removed and the material aspirated. The material was aspirated in a manner to avoid contamination with air, and following successful aspiration, either the syringe was capped and brought immediately to the anaerobic laboratory, or the material was placed in an Anaport bottle.$



Table 2. Patients with Premature Rupture of Membranes and No Infection


Organism Isolated

10 17 22 40 42 45 50 53 54 67

None None None None None S. epidermidis Bifidobacterium None None None

12 14 16 144 2 8 11 27 10 8

hours hours hours hourst hours hours hours hours hours hours

Vaginal Exams P.T.A.* 4 11 6 0 1 0 0 0 0 0

Enterococcus were recovered from the amniotic fluid cultures. Case 2. T h e patient, gravida I, para I, 24 years old, at 36V2 weeks' gestation, had the membranes ruptured to institute labor. Amniocentesis and fluid for culture were obtained 15 hours after rupture of the membranes. Fourteen vaginal examinations had been done prior to amniocentesis. A cesarean section for cephalopelvic disproportion was done. The patient had a febrile postpartum course, and clinical evidence of pneumonia developed. She was treated with ampicillin starting on the first postoperative day. Leukocyte count on admission was normal; it was 17,500 on the second postoperative day, and 14,500 on the third. Blood culture was negative, as was urine culture. The infant had an uneventful course. Peptostreptococcus intermedins was grown from the amniotic fluid.

average of 12.8 vaginal examinations prior to amniocentesis, while those individuals who had premature rupture of the amniotic membranes without evidence of infection had an average of 2.4 vaginal examinations prior to amniocentesis (TaCase 3. The patient was gravida I, para ble 2). A total of 18, or 22%, of women at I, 18 years old, at 43 weeks' gestation. term had prematurely ruptured mem- Amniocentesis was done 13 hours after branes. Eight of these women with prema- rupture of the membranes was done to turely ruptured membranes (44%) had institute labor. Eleven vaginal examinaclinical evidence of infection, and six of tions were done prior to amniocentesis. these (33.3%) had significant organisms The infant did well following delivery. isolated from the amniotic cavity. The mother had a febrile postpartum course, and was thought clinically to have pelvic cellulitis. Blood culture was negaReport of Eight Cases tive; amniotic fluid cultures revealed BacCase 1. The patient was gravida I, para teroides species and Enterococcus species. Case 4. A 17-year-old girl, gravida I, I, 19 years old, at 38 weeks' gestation. Amniocentesis was performed 28 hours para 0, was thought to be at 40 weeks' after spontaneous rupture of membranes. gestation. The amniotic sac was ruptured A total of ten vaginal examinations had 8V2 hours prior to amniocentesis, and an been done. The patient, a chemical dia- internal-monitor catheter was put into betic, became febrile, with a temperature of place. A cesarean section for cephalopel101 F., 6 hours prior to delivery. A febrile vic disproportion was done. The patient postpartum course failed to respond to was febrile postpartum, and she was ampicillin and gentamicin, but responded thought clinically to have endometritis well to Clindomycin. Blood and urine and parametritis. The infant showed no cultures were negative. The infant had an complication. Clindamycin and gentamiuneventful course except for slightly ele- cin appeared to be successful in treating vated bilirubin. Bacteroides ruminicola and the clinical evidence of infection. Lac-

Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016

* I'.T .A. = prior to amniocentesis. tNol included in calculations.

Time Ruptured P.T.A.*

A.J.C.P. —Vol. 65




isolates of Staphylococcus epidennidis, one isolate of an aerobic Corynebacterium sp., and one isolate of a Bifidobacterium sp. The amniotic sac had been previously ruptured in these isolates on only one instance of 5. epidennidis isolation and in the instance of Bifidobacterium. None of these cases was associated with clinical evidence of infection, either in the mother or in the infant. Of 60 patients without significant infection, only nine had premature rupture of the amniotic membranes. Only one of these patients had had more than ten vaginal examinations prior to amniocentesis. This woman had a febrile course, developing a fever of 101.9 F. 6 hours prior to delivery. She was treated empirically with ampicillin and did well. One woman in this group had a Barteroides fragilis septicemia following cesarean section. No organism was grown from the amniotic fluid. It was thought that the septicemia had occurred due to the cesarean section. One patient in this group had a postpartum wound infection which was thought to be unrelated to any pelvic infection. One woman had a febrile course due to pyelonephritis. One other woman had a mild urinary tract infection, post partum, treated with ampicillin. Discussion Harris and Brown, 4 in 1927, studied the bacterial contents of the uterus during cesarean section and concluded that bacterial invasion will increase with vaginal examinations and rupture of membranes. Stroup, 13 in 1962, evaluated amniotic fluid samples in 52 patients. In this study, 44 patients were examined by abdominal puncture, eight by transcervical aspiration. Only two of 44 abdominal samples showed bacterial growth, and the resulting bacteria were considered contami-

Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016

tobacillus species was grown on two occasions, once with the initial amniocentesis, and once from the internal monitor catheter. Case 5. A 23-year-old woman, gravida I, para 0, was at 43 weeks' gestation. Membranes were ruptured 11 Vi hours prior to amniocentesis. The patient had a febrile postpartum course treated with a multitude of antibiotics, including gentamicin, Clindamycin, nitrofurantoin, and cloramphenicol. The infant had no evidence of infection. No anaerobes were grown. Klebsiella species, P. mirabilis E. coli, and Enterococcus were grown. Case 6. A 20-year-old woman, gravida I, para 0, thought to be at 42 weeks' gestation, was admitted for elective induction of labor. The membranes were ruptured 24 hours prior to delivery. She was in labor for a total of 24 hours. Post partum she was thought to have endoparametritis clinically. She was treated with gentamicin and ampicillin. In addition to the E. coli isolated from her amniotic fluid, she had a urinary tract infection due to E. coli. Case 7. A 16-year-old girl, gravida I, was in labor for 32 hours. The membranes were ruptured 5 hours prior to amniocentesis, and 5'/2 hours prior to delivery. The amniotic fluid had a foul odor; however, only S. epidermidis was grown from the amniotic fluid. The patient did well without antibiotic therapy. Case 8. The patient was gravida I, 19 years old, at 40 weeks' gestation. The membranes were ruptured 6 hours prior to delivery. The patient was febrile and manifested abdominal tenderness. She was thought to have endoparametritis and was treated with gentamicin. E. coli was grown both from the amniotic fluid and from the urine. In addition, organisms considered to be contaminants were grown from specimens from eight patients. These included six




fragilis isolated from the transcervical local culture, but the endometrium cultures were sterile. This patient had a transient fever, was treated with antibiotics, and was discharged on her fourth hospital day. All other isolates were considered contaminants. Larsen and associates7 felt that they could predict clinically significant infection by the presence of polymorphonuclear leukocytes in the amniotic fluid, but anaerobic studies were not done. Chow 2 reported 94 cases of neonatal bacteremia, 26 of. which were due to anaerobes. A review of the cases showed that prolonged rupture of the membrane, 24 hours or longer, was the most frequent common denominator. Most strikingly, the bacteriologic findings and neonatal courses of these infants, with one exception, were surprisingly uneventful. The mothers had stable postpartum courses, in general. Chow stated that once the neonates were removed from the infected amniotic fluid, their bacteremia was relatively transient, and that in women with postpartum sepsis, removal of septic foci contributed to their rapid recovery. Our studies would indicate that amniotic fluid from the intact amniotic cavity is sterile. Women who had clinical courses consistent with infection and who had bacteriologic evidence of infection had two characteristics in common. One was premature rupture of the membrane with a rather prolonged course; the other was the number of vaginal examinations done prior to amniocentesis. Morrison, 12 and co-workers 8 found that infection increased following ten or more vaginal examinations prior to delivery. In our study we are not certain whether the large number of vaginal examinations contributed to the infection or were done to follow patients with ruptured membranes who were developing complications, or a combination of the two. In many cases, following rupture of the membranes, amniotic fluid

Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016

nants. Bacterial growth in the transcervical samples was thought to reflect the method of collection rather than infection. It was Stroup's opinion that penetration of intact fetal membranes by vaginal bacteria probably does not occur often, even during labor. Harwick, Iupea, and Fekerty 5 studied amniotic fluid specimens from 60 patients, 44 of whom had been hospitalized for therapeutic abortions at 14 to 20 weeks. This population limited the scope of the patients studied. Of the 60 patients, 44 had sterile amniotic fluid, four cultures yielded Mycoplasma hominis, two cultures were positive for Staphylococcus albus. They noted that Mycoplasma has been associated with spontaneous abortion, and felt that vaginal examination may allow entry of the organism when it is present in the cervix, possibly permitting intrauterine infection in an otherwise normal pregnancy. Prevedourakis and associates10 studied 39 samples of amniotic fluid during pregnancy, and 78 samples obtained during labor by abdominal amniocentesis. Once during pregnancy and eight times d u r i n g labor, organisms thought by the authors to be pathogenic were isolated in the amniotic fluid. No clinical symptoms were found in the mother or in the newborn infant. A total incidence of 7.69% of bacterial invasion in the amniotic cavity was noted. In a further expansion of these studies, apparently using some of the same information and recorded in 1971, Prevedourakis and colleagues 9 found an isolation rate of 7.85% without maternal or fetal morbidity. Spore and co-workers 12 studied the bacteriology of postpartum oviducts and endometrium: 26 women were studied by transuterine aspiration, 5 to 18 hours post partum. In one patient Clostridium perfringens, Bacteroides fragilis, and a Peptostreptococcus were isolated from the endometrium. This patient's postpartum course was unremarkable. One patient had Peptostreptococcus sp. and Bacteroides

A.J.C.P. —Vol. 65

January 1976


anaerobic bacteria be available. We have also demonstrated that fluid removed from the intact amniotic cavity is sterile for aerobic, facultative anaerobic, and anaerobic bacteria. References 1. Blair JE, Lennette EH, Truant JP: Manual of Clinical Microbiology. American Society Microbiologists, 1970 2. Chow AW: When to suspect congenital anaerobic infection. Hosp Pract 9:59, 1974 3. Gorbach SL, Menda KB, Thadepalli H, et al: Anaerobic microflora of the cervix in healthy women. Am J Obstet Gynecol 117:1053— 1055, 1973 4. Harris JW, Brown H: T h e bacterial content of the uterus at caesarean section. Am J Obstet Gynecol 13:133-143, 1972 5. Harwick JF, Iuppa J B , Fekerty FR: Microorganisms and amniotic fluid. Obstet Gynecol 33:356-359, 1969 6. Holdeman LV, Moore WEC: Anaerobe Laboratory Manual. Second edition. Virginia Polytechnic Institute Anaerobe Laboratory, 1973 7. Larsen JW, Goldkrand JW, Hanson TM, et al: Intrauterine infection on an obstetric service. Obstet Gynecol Obser 12:4, July 1973 8. Morrison JC, Coxwell WL, Kennedy BS, et al: T h e use of prophylactic antibiotics in patients undergoing caesarean section. Surg Gynecol Obstet 136:425-428, 1973 9. Prevedourakis CN, Strigou-Charlabis E, Kaskarelis CB: Bacterial invasion of amniotic cavity during pregnancy and labor. Obstet Gynecol 37:459-461, 1971 10. Prevedourakis G, Papadimitriou G, Ioannidou A: Isolation of pathogenic bacteria in the amniotic fluid during pregnancy and labor. Am J Obstet Gynecol 106:400-402, 1970 11. Sharpe ME, Hill LR, Lapage SP: Pathogenic lactobacilli. J Med Microbiol 6:281-286, 1973 12. Spore WW, Moskal PA, Nakamura RM, et al: Bacteriology of post partum oviducts and endometrium. Am J Obstet Gynecol 7:572577, 1970 13. Stroup PE: Amniotic fluid infection and the intact fetal m e m b r a n e . Obstet Gynecol 19:736-739, 1962 14. Sweet RL, Ledger WJ: Puerperal infectious morbidity. Am J Obstet Gynecol 117:10931100, 1973 15. Thadepalli H, Gorbach SL, Keith L: Anaerobic infections of the female genital tract: Bacteriologic and therapeutic aspects. Am J Obstet Gynecol 117:1034-1040, 1973

Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016

is difficult to obtain by transabdominal aspiration due to loss of fluid. As use of internal catheter monitors becomes more prevalent, we suspect that it may be a satisfactory method for obtaining the material for culture. More experience will have to be gained with this method in order to evaluate the presence of "normal flora," such as those in our case of Lactobacillus, within the amniotic cavity. One hundred per cent of the patients with infection had ruptured membranes, while only 15% of the unaffected individuals had ruptured membranes. The average number of vaginal examinations in patients with infections was 13.7, while that for those with ruptured membranes and without infection was 2.8. All patients who had significant bacteriologic findings in the amniotic fluid were at or near term. In addition, four patients who were undergoing voluntary interruption of pregnancy developed clinical evidence of infection; however, no organisms were identified. In these four women, there was no evidence of pulmonary or urinary tract infection, so we assume that these were pelvic infections. Additional studies are under way at this time to evaluate bacteriologically the nature of the inflammatory response seen in some of these patients undergoing voluntary interruption of pregnancy. Based on previous studies and our findings, we suggest that amniocentesis for culture be when amniotic membranes have been ruptured for more than 8 hours, especially when seven or more vaginal examinations have been done. Our experience and that of others 3,14,15 would indicate that in order to obtain optimal information, adequate anaerobic collection technics be used, and adequate methods for isolation and identification of


Evaluation of amniotic fluid for aerobic and anaerobic bacteria.

Studies of 117 pregnant women, 83 at term, were instituted to determine the bacteriologic state of amniotic fluid, utilizing both standard aerobic and...
433KB Sizes 0 Downloads 0 Views