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Transmission of Citrobacter koseri from mother to infant documented by ribotyping and pulsed-field gel electrophoresis
ELSEVIER
Transmission of Citrobacter koseri from Mother to Infant Documented by Ribotyping and Pulsed-Field Gel Electrophoresis Christopher J. Papasian, Janet Kinney, Stacy Coffman, Richard J. Hollis, and Michael A. Pfaller
We describe a case in which Citrobacter koseri (formerly C. diversus) was transmitted from a pregnant mother with chorioamnionitis and bacteremia to her infant who was bacferemic at birth and in apparent septic shock. Two highly discriminating molecular methods, ribotyping and pulsed field gel elecfrophoresis, were used to examine restriction fragment length
INTRODUCTION koseri (formerly C. diversus) is a rare but devastating neonatal pathogen because of its propensity to produce leptomeningitis that often progresses to brain abscess (Lin et al. 1987). Infants developing C. koseri meningitis have a 77% chance of developing a brain abscess and a mortality rate of 34%; 90% of survivors suffer mental retardation (Harvey et al. 1995). Several clusters of neonatal C. koseri infections have been reported (Harvey et al. 1995; Williams et al. 1984). These clusters usually last several months to years, produce relatively few cases, and the implicated strain is often recovered from many asympCitrobacter
polymorphisms within the genomic DNA of maternal and infant isolates. Both techniques identified the maternal and infant isolates as the same strain, distinct from epidemiologically unrelated controls, thus confirming their common origin. 0 1996 Elsevier Science Inc.
tomatic infants. For every infant developing invasive disease there are 6 to 20 asymptomatic infants colonized with C. koseri (Harvey et al. 1995; Morris et al. 1986). The manner in which C. koseri initially enters the nursery is often unclear. Several studies have implicated mothers of infants as a possible source; however, this method of introduction is not universally accepted (Lin et al. 1987). We report a case in which C. koseri was transmitted vertically from mother to infant, both of whom were bacteremic. Identity of maternal and infant isolates was supported by molecular analysis.
CASE From the Department of Pathology, (CJP, JK) Truman Medical Center University of Missouri, School of Medicine; Kansas City, Missouri, the Department of Pediatrics, fJK) Children’s Mercy Hospital and the Department of Pathology, (SC, RJH, MAP) University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA. Address reprint requests to: Christopher J. Papasian, Ph.D., Director, Diagnostic Microbiology & Immunology, Truman Medical Center, 2301 Holmes Street, Kansas City, MO 64108, USA. Received 17 June 1996; revised and accepted 17 September 1996.
DIAGN MICROBIOL INFECT DIS 1996;26:63-67 0 1996 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
REPORT
A 34-year-old woman presented to obstetrics clinic at 35 weeks gestation for suspected cervical contractions. She had three healthy children (all delivered by Caesarian section) and two spontaneous miscarriages in her five previous pregnancies; her current pregnancy was complicated by polyhydramnios. Prenatal screening tests for Chlnmydia frachomafis, Neisseria gonorrhoea, Group B Streptococcus (GBS), and
073X3893/96/$15.00 PI1 SO732-8893(96)00177-O
64
hepatitis B surface antigen were negative; a Pap smear was within normal limits, and rubella serology indicated immunity. The patient was stable and afebrile, and fetal heart tones were 140 to 150/min and reactive. The patient wanted to have a vaginal delivery, and she remained hospitalized for expectant management. She experienced spontaneous rupture of amniotic membranes (ROM), discharging clear fluid, approximately 19 hours after initial presentation. Repeat GBS cultures were performed within 1 hour of ROM, and intravenous ampicillin therapy (2 g every 6 h) was initiated 7 hours after ROM; ampicillin therapy was discontinued approximately 48 hours later because of negative GBS cultures. Fetal lung maturity studies done 3 days after ROM indicated equivocal lung maturity (Lecithin/ Sphingomyelin ratio, 2.2; phosphatidyl glycerol, negative). Seven days after ROM, the patient began having irregular contractions at 3 to 4 minute intervals. Fetal heart tones were 160 to 170/min, and induction of labor with pitocin was attempted. The patient because febrile approximately 4 hours after contractions were first noted [temperature, 100.5”F (38.1”01, and fetal heart tones were 180 to 190/min; internal monitors were placed and amnioinfusion was begun. Intravenous therapy with ampicillin (2 g every 6 h) and gentamicin (100 mg every 8 h) was initiated. An hour later, her rectal temperature was 106°F (41.1”C); two sets of blood cultures (Bactec NR6A, NR7A, NR16A Becton Dickinson Microbiology Systems, Sparks, MD, USA) were collected, and a peripheral white blood cell (WBC) count revealed 21,200 WBC/mm3 (56% polymorphonuclear neutrophils, 38% band cells, 4% lymphocytes and 2% monocytes). Pitocin induction was discontinued because of fetal tachycardia and persistent decelerations, and a low transverse Caesarean section was performed; a 2525-g female infant was delivered with APGARs of 3 and 7. The placenta appeared infected, and swab specimens were collected for bacterial culture. The mother had a brief hypotensive episode postoperatively, and intravenous clindamytin (900 mg every 8 h) was added to her therapeutic regimen. Cifrobacfer kosevi was recovered from the placenta and from both sets of blood cultures (an aerobic bottle from one set, and an aerobic and resincontaining bottle from the second set); the identification (API 20E; bioMerieux Vitek, In., Hazelwood, MO, USA and Neg Combo Panel #13; Dade Microscan, West Sacramento, CA, USA) and susceptibility for all isolates were reported 3 days postoperatively. All isolates were susceptible (Neg Combo Panel #13; Dade Microscan) to gentamicin, tobramycin, piperacillin, ampicillin-sulbactam, ticarcillin-clavulanate,
C.J. Papasian et al.
cefazolin, cefotetan, ceftriaxone, ceftazidime, aztreonam, imipenem, ciprofloxacin and trimethroprimsulfamethoxazole. The isolates had an intermediate susceptibility to ampicillin (MIC = 16 +g/ml). Ampicillin and clindamycin were discontinued, and piperacillin (3 g intravenously every 6 h) was added shortly after susceptibility results were reported. Additionally, the gentamicin dosage was increased to 125 mg every 8 h based on peak gentamicin levels of 4.1 to 4.4 kg/ml on the previous dosage regimen; the mother was febrile [temp, 103.2”F (39.6”C)J at this time and had a peripheral WBC count of 19,700/ mm3 (74% polys, 13% bands 10% lymphocytes, 2% monocytes, and 1% metamyelocytes). The mother gradually defervesced and was afebrile 7 days postoperatively. Her intravenous antibiotics were discontinued, and she was discharged the following day but remained on oral cephalexin therapy (500 mg every 6 h) for 9 additional days. The infant was a 35-week, 2525 gram, average for gestational age female who appeared ill at delivery, with tachycardia, poor perfusion, and hypertonia. She was taken immediately to the intensive care nursery for respiratory distress and probable septic shock. The physical examination was remarkable for tachypnea, intercostal retractions, and tachycardia without murmur. Extremities showed acrocyanosis with prolonged capillary refill and weak femoral pulses. Neurologically, the infant was awake, but hypotonic and lethargic. Shortly after birth, roentgenograms of the chest showed hazy lung fields consistent with respiratory distress syndrome. The infant’s initial peripheral WBC count was 13,200/mm3 (12% polymorphonuclear neutrophils, 10% bands, 69% lymphocytes, 5% monocytes, 3% eosinophils, 1 metamyelocyte). Hemoglobin, hematocrit, and platelet count were 12.2 g/dl, 36.3%, and 252,00O/mm”, respectively, and numerous (57%) nucleated erythrocytes were noted on the peripheral smear. The initial arterial blood gas showed: pH, 7.07, pCO,, 67 mm Hg; PO,, 94 mm Hg; HCO,, 19.5 mmol/l. The infant was treated with sodium bicarbonate, dopamine, ampicillin (100 mg/kg every 12 h iv), and gentamicin (25 mg/kg every 12 h iv). On the 2nd day of life, her blood culture was positive for Gram-negative rods, which were subsequently identified as C. kosevi. Antibiotic therapy was changed to cefotaxime (50 mg/kg every 12 h iv) and gentamicin for a total 14-day course. The following day, a lumbar puncture revealed a slightly cloudy CSF with erythrocyte and WBC counts of 4,000/mm3 and 2/mm3 (9% polymorphonuclear neutrophils, 91% mononuclear cells), respectively. Cerebrospinal fluid glucose and CSF protein were 59 mg/dl and 145 mg/dl, respectively, but bacterial cultures Jtryp-
Vertical
Citrobacter
koseri Transmission
65
ticase soy agar with 5% sheep erythrocytes, chocolate agar, and eugenic broth (Remel, Lenexa, KS, USA)] were negative. Computerized axial tomograms of the head, with and without contrast, were normal on days 3 and 10 of life. At discharge, the patient was neurologically stable, and her physical examination was unremarkable; she was seen in clinic at followup and was felt to be developing and growing well. Bloodstream isolates of C. koseri from the mother and infant were typed using ribotyping and pulsedfield gel electrophoresis (PFGE) techniques. The profiles were compared with two epidemiologically unrelated isolates of C. koseri. Ribotyping was performed using the restriction enzyme Eco RI and the RiboPrinter (Wilmington, DE, USA) automated ribotyping system (Pfaller et al. 1996). Pulsed-field gel electrophoresis typing was performed using restriction enzymes Spe I and Xba Z followed by electrophoresis using a CHEF DRII system (BioRad) as described previously (Pfaller et al. 1994). The results of the molecular typing studies revealed that the isolates of C. koseri from the mother and infant were identical by both PFGE (Figure 1) and ribotyping techniques (data not shown) and were distinctly different from the epidemiologically unrelated isolates.
01234D5678D
kb 242.5 kb
145.5 kb
49.5 kLb
FIGURE
1.
Pulsed-field
gel electrophoresis
patterns
of
Citrobacter koseri obtained using the restriction enzymes Spe I (lanes 1 to 4) and Xba I (lanes 5 to 8). Lane D contains lambda ladder molecular mass standards (in kilobases). Lanes 1 and 2 (Spe 0 and 5 and 6 (Xba 0 represent two epidemiologically unrelated strains of C. koseri. Lanes 3 and 4 (Spe I) and 7 and 8 (Xba I) represent isolates obtained from the mother (lanes 3 and 7) and infant (lanes 4 and 8).
DISCUSSION Vertical transmission of C. koseri has been documented on six previous occasions (Table 1); maternal and infant isolates were compared using molecular methods in four of these cases. Finn et al. (1988) report the case of a 1670-g girl born at 34 weeks of gestation to a 26-year-old woman 11 hours after ROM. The mother had a past history of spontaneous abortions, and a cervical cerclage suture was inserted at 20 weeks of gestation; the suture was removed before induction of labor and subsequently grew C. koseri. The mother remained asymptomatic and was not treated with antibiotics, but the infant developed abdominal distension and became lethargic on the 5th day of life; C. koseri was recovered from the baby’s blood, umbilicus, and rectum. Examination of cerebrospinal fluid was consistent with bacterial meningitis, but cultures were negative. The infant responded to treatment with gentamicin and cefotaxime. Plasmid profile analysis confirmed the identity of maternal and infant isolates. In the second case, a 3800-g male in fetal distress was delivered by cesarean section to a 21-year-old (Harvey et al. 1995). The mother had spontaneous ROM 30 hours before delivery and developed fever to 1Ol’F (38.3”C); she was treated with ampicillin, gentamicin, and clindamycin for presumed chorioamnionitis. The infant required resuscitation immediately after delivery, and cranial ultrasonography performed on the 2nd day of life showed possible intraventricuar hemorrhages; blood and tracheal aspirate cultures collected on day 1 grew C. koseri. The infant was treated with cefotaxime for 21 days for sepsis and presumed meningitis (CSF collected on day 16 was culture negative, but 29 leukocytes were present); at age 12 months, the infant had global developmental delay, hypertonicity, and microcephaly. The mother remained febrile on antibiotics for 1 week, but was clinically well after a 16-day postpartum course of antibiotics; C. koseri was recovered from amniotic infusion fluid and maternal blood cultures collected 29 hours postpartum. Pathologic examination of the placenta revealed funisitis and chorioamnionitis. Identity of maternal and infant isolates was confirmed by analysis of DNA fingerprints generated by repetitive element based polymerase chain reaction. The two other reports of vertical C. koseri transmission documented by molecular methods were identified by surveillance programs initiated because of clusters of C. koseri infection; neither mother nor infant were symptomatic in either case (Morris et al. 1986, Williams et al. 1984). There are two additional cases of symptomatic babies born to mothers colonized or infected with C.
C.J. Papasian et al.
TABLE
1
Comparison of Current Case with Previously Vertical Transmission Source of Isolate
Case
Mother
Documented
Cases of C. koseri (diversus)
Clinical Manifestations Infant
Mother
Infant
Unknown
Unknown
Asymptomatic
Asymptomatic
Unknown
Unknown
Asymptomatic
Asymptomatic
Cervix, placenta
Blood, nose, ear, throat
Asymptomatic
Sepsis, death
Placenta
Blood, tracheal aspirate
Chorioamnionitis
Sepsis, possible meningitis
Cervical cerclage Suture
Blood, umbilicus, rectal swab
Asymptomatic
Sepsis, possible meningitis
Blood, amniotic fluid
Blood, tracheal aspirate
Chorioamnionitis
Sepsis, possible meningitis
Blood, placenta
Blood
Chorioamnionitis sepsis
Sepsis
Isolate Characterization Plasmid profile, biotype, serotype, chromosomal restriction endonuclease digest Plasmid profile, serotype Biotype, antimicrobial susceptibility Biotype, antimicrobial susceptibility Plasmid profile analysis, biotype, antimicrobial susceptibility Repetitive element PCR analysis Ribotyping, pulsed-field
Reference Morris et al. 1986
Williams et al. 1984 Dan and Sadan 1989 Walter 1988
Finn et al. 1988
Harvey et al. 1995 Current case
gel electrophoresis
koseri. The maternal/infant strain identity in these two cases, however, is based only on similar biotypes and antimicrobial susceptibility patterns; no molecular studies were performed. In the first case, a 36year-old woman was hospitalized for premature ROM at 29 weeks of gestation (Dan and Sadan 1989). She was treated with ampicillin and remained afebrile; however, she developed tachycardia and leukocytosis so gentamicin was added. A cerclage suture (inserted at 14 weeks gestation) was removed, and labor was induced. A 985-g girl was delivered 36 hours after ROM. The infant became septic and died the next day; C. koseri was recovered from the nose, ear, throat, and blood. Citrobncter koseri was also recovered from the mother’s cervix and placenta, but she remained afebrile and well throughout the postpartum period. In the second case, an 800-g female dizygotic twin was delivered at 27 weeks of gestation to a 29-year-old 18 hours after ROM (Walter 1988). The mother was admitted 2 weeks earlier because of preterm labor, leukocytosis, and a low grade fever; she received ampicillin and apparently defervesced. The mother developed fever, abdominal pain, and leukocytosis 1 hour before delivery and had purulent, foul smelling amniotic fluid; C. koseri was re-
covered from the placenta, and pathologic examination revealed chorioamnionitis. The infant was leukocytotic with a left shift, and C. koseri was recovered from blood and an endotracheal aspirate; CSF obtained after 24 hours of therapy with cefotaxime and gentamicin showed pleocytosis and hypoglycorrachia, but cultures were negative. The infant received antibiotics for 3 weeks and recovered uneventfully. We report the third case of vertical C. koseri transmission to a symptomatic infant in which identity of maternal and infant isolates is supported by molecular analysis. The case is notable because the mother had ROM for 7 days prior to delivery and developed chorioamnionitis and C. koseri bacteremia. The infant had profound metabolic acidosis, was neurologically depressed, in septic shock, and bacteremic with C. koseri at delivery, indicating in utero infection. We used two highly discriminating methods, ribotyping and PFGE, to examine restriction fragment length polymorphisms within the genomic DNA of maternal and infant isolates. Both techniques identified the maternal and infant isolates as the same strain, distinct from epidemiologically unrelated controls, thus confirming their common origin.
Vertical
67
Citrobnctev koseri Transmission
REFERENCES Dan M, Sadan 0 (1989) VerticaI transmission of Citrobacter diversus from mother to infant. Ped Infect Dis J 8:63-64. Lin FC, Devoe WF, Morrison C, Libonati J, Powers I’, Gross RJ, Rowe 8, Israel E, Morris JG Jr. (1987) Outbreak of neonatal Citrobacfer diversus meningitis in a suburban hospital. Ped Infect Dis J 6:50-55. Finn A, Talbot GH, Anday E, Skros M, Provencher M, and Hoegg C (1988) Vertical transmission of Citrobacter diversus from mother to infant. Ped Infect Dis J 71293-294. Harvey BS, Koeuth T, Versalovic J, Woods CR, Lupski JR (1995) Vertical transmission of Citvobacter diversus documented by DNA fingerprinting. Infect Conf Hosp Epidemiol 16:564-569. Morris JG Jr, Feng-Ying CL, Morrison CB, Gross RJ, Khabbaz R, Maher KOD, Rowe B, Israel E, Libonati J (1986) Molecular epidemiology of neonatal meningitis due to Citrobacter diversus: A study of isolates from hospitals in Maryland. 1 Infect Dis 154:409414. Pfaller MA, Hollis RJ, Sader HS (1994) Chromosomal
re-
striction fragment analysis by pulsed-field gel electrophoresis. In Ed Clinical Microbiology Procedures Handbook (suppl 1) HD Isenberg. Washington, DC: American Society for Microbiology, p. 10.5C.12. Pfaller MA, Wendt C, Hollis RJ, Wenzel RI’, Fritschel SJ, Neubauer JJ, Herwaldt LA (1996) Comparative evaluation of an automated ribotyping system versus pulsedfield gel electrophoresis for epidemiological typing of clinical isolates of Escherichia coli and Pseudomonas aeruginosa from patients with recurrent Gram-negative bacteremia. Diagn Microbial Infect Dis submitted for publication. Walter E (1988) Vertical transmission of Citrobacter diversus from mother to infant. Ped Infect Dis J 7~675. Williams WW, Mariano J, Spurrier M, Donnell HD Jr, Breckenridge RL Jr, Anderson RL, Wachsmuth IK, Thornsberry C, Graham DR, Thibeault DW, Allen JR (1984) Nosocomial meningitis due to Citrobacter diversus in neonates; New aspects of the epidemiology. I Infect Dis 150~229-235.
Transmission of Citrobacter koseri from Mother to Infant Documented by Ribotyping and Pulsed-Field Gel Electrophoresis Christopher J. Papasian, Janet Kinney, Stacy Coffman, Richard J. Hollis, and Michael A. Pfaller
We describe a case in which Citrobacter koseri (formerly C. diversus) was transmitted from a pregnant mother with chorioamnionitis and bacteremia to her infant who was bacferemic at birth and in apparent septic shock. Two highly discriminating molecular methods, ribotyping and pulsed field gel elecfrophoresis, were used to examine restriction fragment length
INTRODUCTION koseri (formerly C. diversus) is a rare but devastating neonatal pathogen because of its propensity to produce leptomeningitis that often progresses to brain abscess (Lin et al. 1987). Infants developing C. koseri meningitis have a 77% chance of developing a brain abscess and a mortality rate of 34%; 90% of survivors suffer mental retardation (Harvey et al. 1995). Several clusters of neonatal C. koseri infections have been reported (Harvey et al. 1995; Williams et al. 1984). These clusters usually last several months to years, produce relatively few cases, and the implicated strain is often recovered from many asympCitrobacter
polymorphisms within the genomic DNA of maternal and infant isolates. Both techniques identified the maternal and infant isolates as the same strain, distinct from epidemiologically unrelated controls, thus confirming their common origin. 0 1996 Elsevier Science Inc.
tomatic infants. For every infant developing invasive disease there are 6 to 20 asymptomatic infants colonized with C. koseri (Harvey et al. 1995; Morris et al. 1986). The manner in which C. koseri initially enters the nursery is often unclear. Several studies have implicated mothers of infants as a possible source; however, this method of introduction is not universally accepted (Lin et al. 1987). We report a case in which C. koseri was transmitted vertically from mother to infant, both of whom were bacteremic. Identity of maternal and infant isolates was supported by molecular analysis.
CASE From the Department of Pathology, (CJP, JK) Truman Medical Center University of Missouri, School of Medicine; Kansas City, Missouri, the Department of Pediatrics, fJK) Children’s Mercy Hospital and the Department of Pathology, (SC, RJH, MAP) University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA. Address reprint requests to: Christopher J. Papasian, Ph.D., Director, Diagnostic Microbiology & Immunology, Truman Medical Center, 2301 Holmes Street, Kansas City, MO 64108, USA. Received 17 June 1996; revised and accepted 17 September 1996.
DIAGN MICROBIOL INFECT DIS 1996;26:63-67 0 1996 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
REPORT
A 34-year-old woman presented to obstetrics clinic at 35 weeks gestation for suspected cervical contractions. She had three healthy children (all delivered by Caesarian section) and two spontaneous miscarriages in her five previous pregnancies; her current pregnancy was complicated by polyhydramnios. Prenatal screening tests for Chlnmydia frachomafis, Neisseria gonorrhoea, Group B Streptococcus (GBS), and
073X3893/96/$15.00 PI1 SO732-8893(96)00177-O
64
hepatitis B surface antigen were negative; a Pap smear was within normal limits, and rubella serology indicated immunity. The patient was stable and afebrile, and fetal heart tones were 140 to 150/min and reactive. The patient wanted to have a vaginal delivery, and she remained hospitalized for expectant management. She experienced spontaneous rupture of amniotic membranes (ROM), discharging clear fluid, approximately 19 hours after initial presentation. Repeat GBS cultures were performed within 1 hour of ROM, and intravenous ampicillin therapy (2 g every 6 h) was initiated 7 hours after ROM; ampicillin therapy was discontinued approximately 48 hours later because of negative GBS cultures. Fetal lung maturity studies done 3 days after ROM indicated equivocal lung maturity (Lecithin/ Sphingomyelin ratio, 2.2; phosphatidyl glycerol, negative). Seven days after ROM, the patient began having irregular contractions at 3 to 4 minute intervals. Fetal heart tones were 160 to 170/min, and induction of labor with pitocin was attempted. The patient because febrile approximately 4 hours after contractions were first noted [temperature, 100.5”F (38.1”01, and fetal heart tones were 180 to 190/min; internal monitors were placed and amnioinfusion was begun. Intravenous therapy with ampicillin (2 g every 6 h) and gentamicin (100 mg every 8 h) was initiated. An hour later, her rectal temperature was 106°F (41.1”C); two sets of blood cultures (Bactec NR6A, NR7A, NR16A Becton Dickinson Microbiology Systems, Sparks, MD, USA) were collected, and a peripheral white blood cell (WBC) count revealed 21,200 WBC/mm3 (56% polymorphonuclear neutrophils, 38% band cells, 4% lymphocytes and 2% monocytes). Pitocin induction was discontinued because of fetal tachycardia and persistent decelerations, and a low transverse Caesarean section was performed; a 2525-g female infant was delivered with APGARs of 3 and 7. The placenta appeared infected, and swab specimens were collected for bacterial culture. The mother had a brief hypotensive episode postoperatively, and intravenous clindamytin (900 mg every 8 h) was added to her therapeutic regimen. Cifrobacfer kosevi was recovered from the placenta and from both sets of blood cultures (an aerobic bottle from one set, and an aerobic and resincontaining bottle from the second set); the identification (API 20E; bioMerieux Vitek, In., Hazelwood, MO, USA and Neg Combo Panel #13; Dade Microscan, West Sacramento, CA, USA) and susceptibility for all isolates were reported 3 days postoperatively. All isolates were susceptible (Neg Combo Panel #13; Dade Microscan) to gentamicin, tobramycin, piperacillin, ampicillin-sulbactam, ticarcillin-clavulanate,
C.J. Papasian et al.
cefazolin, cefotetan, ceftriaxone, ceftazidime, aztreonam, imipenem, ciprofloxacin and trimethroprimsulfamethoxazole. The isolates had an intermediate susceptibility to ampicillin (MIC = 16 +g/ml). Ampicillin and clindamycin were discontinued, and piperacillin (3 g intravenously every 6 h) was added shortly after susceptibility results were reported. Additionally, the gentamicin dosage was increased to 125 mg every 8 h based on peak gentamicin levels of 4.1 to 4.4 kg/ml on the previous dosage regimen; the mother was febrile [temp, 103.2”F (39.6”C)J at this time and had a peripheral WBC count of 19,700/ mm3 (74% polys, 13% bands 10% lymphocytes, 2% monocytes, and 1% metamyelocytes). The mother gradually defervesced and was afebrile 7 days postoperatively. Her intravenous antibiotics were discontinued, and she was discharged the following day but remained on oral cephalexin therapy (500 mg every 6 h) for 9 additional days. The infant was a 35-week, 2525 gram, average for gestational age female who appeared ill at delivery, with tachycardia, poor perfusion, and hypertonia. She was taken immediately to the intensive care nursery for respiratory distress and probable septic shock. The physical examination was remarkable for tachypnea, intercostal retractions, and tachycardia without murmur. Extremities showed acrocyanosis with prolonged capillary refill and weak femoral pulses. Neurologically, the infant was awake, but hypotonic and lethargic. Shortly after birth, roentgenograms of the chest showed hazy lung fields consistent with respiratory distress syndrome. The infant’s initial peripheral WBC count was 13,200/mm3 (12% polymorphonuclear neutrophils, 10% bands, 69% lymphocytes, 5% monocytes, 3% eosinophils, 1 metamyelocyte). Hemoglobin, hematocrit, and platelet count were 12.2 g/dl, 36.3%, and 252,00O/mm”, respectively, and numerous (57%) nucleated erythrocytes were noted on the peripheral smear. The initial arterial blood gas showed: pH, 7.07, pCO,, 67 mm Hg; PO,, 94 mm Hg; HCO,, 19.5 mmol/l. The infant was treated with sodium bicarbonate, dopamine, ampicillin (100 mg/kg every 12 h iv), and gentamicin (25 mg/kg every 12 h iv). On the 2nd day of life, her blood culture was positive for Gram-negative rods, which were subsequently identified as C. kosevi. Antibiotic therapy was changed to cefotaxime (50 mg/kg every 12 h iv) and gentamicin for a total 14-day course. The following day, a lumbar puncture revealed a slightly cloudy CSF with erythrocyte and WBC counts of 4,000/mm3 and 2/mm3 (9% polymorphonuclear neutrophils, 91% mononuclear cells), respectively. Cerebrospinal fluid glucose and CSF protein were 59 mg/dl and 145 mg/dl, respectively, but bacterial cultures Jtryp-
Vertical
Citrobacter
koseri Transmission
65
ticase soy agar with 5% sheep erythrocytes, chocolate agar, and eugenic broth (Remel, Lenexa, KS, USA)] were negative. Computerized axial tomograms of the head, with and without contrast, were normal on days 3 and 10 of life. At discharge, the patient was neurologically stable, and her physical examination was unremarkable; she was seen in clinic at followup and was felt to be developing and growing well. Bloodstream isolates of C. koseri from the mother and infant were typed using ribotyping and pulsedfield gel electrophoresis (PFGE) techniques. The profiles were compared with two epidemiologically unrelated isolates of C. koseri. Ribotyping was performed using the restriction enzyme Eco RI and the RiboPrinter (Wilmington, DE, USA) automated ribotyping system (Pfaller et al. 1996). Pulsed-field gel electrophoresis typing was performed using restriction enzymes Spe I and Xba Z followed by electrophoresis using a CHEF DRII system (BioRad) as described previously (Pfaller et al. 1994). The results of the molecular typing studies revealed that the isolates of C. koseri from the mother and infant were identical by both PFGE (Figure 1) and ribotyping techniques (data not shown) and were distinctly different from the epidemiologically unrelated isolates.
01234D5678D
kb 242.5 kb
145.5 kb
49.5 kLb
FIGURE
1.
Pulsed-field
gel electrophoresis
patterns
of
Citrobacter koseri obtained using the restriction enzymes Spe I (lanes 1 to 4) and Xba I (lanes 5 to 8). Lane D contains lambda ladder molecular mass standards (in kilobases). Lanes 1 and 2 (Spe 0 and 5 and 6 (Xba 0 represent two epidemiologically unrelated strains of C. koseri. Lanes 3 and 4 (Spe I) and 7 and 8 (Xba I) represent isolates obtained from the mother (lanes 3 and 7) and infant (lanes 4 and 8).
DISCUSSION Vertical transmission of C. koseri has been documented on six previous occasions (Table 1); maternal and infant isolates were compared using molecular methods in four of these cases. Finn et al. (1988) report the case of a 1670-g girl born at 34 weeks of gestation to a 26-year-old woman 11 hours after ROM. The mother had a past history of spontaneous abortions, and a cervical cerclage suture was inserted at 20 weeks of gestation; the suture was removed before induction of labor and subsequently grew C. koseri. The mother remained asymptomatic and was not treated with antibiotics, but the infant developed abdominal distension and became lethargic on the 5th day of life; C. koseri was recovered from the baby’s blood, umbilicus, and rectum. Examination of cerebrospinal fluid was consistent with bacterial meningitis, but cultures were negative. The infant responded to treatment with gentamicin and cefotaxime. Plasmid profile analysis confirmed the identity of maternal and infant isolates. In the second case, a 3800-g male in fetal distress was delivered by cesarean section to a 21-year-old (Harvey et al. 1995). The mother had spontaneous ROM 30 hours before delivery and developed fever to 1Ol’F (38.3”C); she was treated with ampicillin, gentamicin, and clindamycin for presumed chorioamnionitis. The infant required resuscitation immediately after delivery, and cranial ultrasonography performed on the 2nd day of life showed possible intraventricuar hemorrhages; blood and tracheal aspirate cultures collected on day 1 grew C. koseri. The infant was treated with cefotaxime for 21 days for sepsis and presumed meningitis (CSF collected on day 16 was culture negative, but 29 leukocytes were present); at age 12 months, the infant had global developmental delay, hypertonicity, and microcephaly. The mother remained febrile on antibiotics for 1 week, but was clinically well after a 16-day postpartum course of antibiotics; C. koseri was recovered from amniotic infusion fluid and maternal blood cultures collected 29 hours postpartum. Pathologic examination of the placenta revealed funisitis and chorioamnionitis. Identity of maternal and infant isolates was confirmed by analysis of DNA fingerprints generated by repetitive element based polymerase chain reaction. The two other reports of vertical C. koseri transmission documented by molecular methods were identified by surveillance programs initiated because of clusters of C. koseri infection; neither mother nor infant were symptomatic in either case (Morris et al. 1986, Williams et al. 1984). There are two additional cases of symptomatic babies born to mothers colonized or infected with C.
C.J. Papasian et al.
TABLE
1
Comparison of Current Case with Previously Vertical Transmission Source of Isolate
Case
Mother
Documented
Cases of C. koseri (diversus)
Clinical Manifestations Infant
Mother
Infant
Unknown
Unknown
Asymptomatic
Asymptomatic
Unknown
Unknown
Asymptomatic
Asymptomatic
Cervix, placenta
Blood, nose, ear, throat
Asymptomatic
Sepsis, death
Placenta
Blood, tracheal aspirate
Chorioamnionitis
Sepsis, possible meningitis
Cervical cerclage Suture
Blood, umbilicus, rectal swab
Asymptomatic
Sepsis, possible meningitis
Blood, amniotic fluid
Blood, tracheal aspirate
Chorioamnionitis
Sepsis, possible meningitis
Blood, placenta
Blood
Chorioamnionitis sepsis
Sepsis
Isolate Characterization Plasmid profile, biotype, serotype, chromosomal restriction endonuclease digest Plasmid profile, serotype Biotype, antimicrobial susceptibility Biotype, antimicrobial susceptibility Plasmid profile analysis, biotype, antimicrobial susceptibility Repetitive element PCR analysis Ribotyping, pulsed-field
Reference Morris et al. 1986
Williams et al. 1984 Dan and Sadan 1989 Walter 1988
Finn et al. 1988
Harvey et al. 1995 Current case
gel electrophoresis
koseri. The maternal/infant strain identity in these two cases, however, is based only on similar biotypes and antimicrobial susceptibility patterns; no molecular studies were performed. In the first case, a 36year-old woman was hospitalized for premature ROM at 29 weeks of gestation (Dan and Sadan 1989). She was treated with ampicillin and remained afebrile; however, she developed tachycardia and leukocytosis so gentamicin was added. A cerclage suture (inserted at 14 weeks gestation) was removed, and labor was induced. A 985-g girl was delivered 36 hours after ROM. The infant became septic and died the next day; C. koseri was recovered from the nose, ear, throat, and blood. Citrobncter koseri was also recovered from the mother’s cervix and placenta, but she remained afebrile and well throughout the postpartum period. In the second case, an 800-g female dizygotic twin was delivered at 27 weeks of gestation to a 29-year-old 18 hours after ROM (Walter 1988). The mother was admitted 2 weeks earlier because of preterm labor, leukocytosis, and a low grade fever; she received ampicillin and apparently defervesced. The mother developed fever, abdominal pain, and leukocytosis 1 hour before delivery and had purulent, foul smelling amniotic fluid; C. koseri was re-
covered from the placenta, and pathologic examination revealed chorioamnionitis. The infant was leukocytotic with a left shift, and C. koseri was recovered from blood and an endotracheal aspirate; CSF obtained after 24 hours of therapy with cefotaxime and gentamicin showed pleocytosis and hypoglycorrachia, but cultures were negative. The infant received antibiotics for 3 weeks and recovered uneventfully. We report the third case of vertical C. koseri transmission to a symptomatic infant in which identity of maternal and infant isolates is supported by molecular analysis. The case is notable because the mother had ROM for 7 days prior to delivery and developed chorioamnionitis and C. koseri bacteremia. The infant had profound metabolic acidosis, was neurologically depressed, in septic shock, and bacteremic with C. koseri at delivery, indicating in utero infection. We used two highly discriminating methods, ribotyping and PFGE, to examine restriction fragment length polymorphisms within the genomic DNA of maternal and infant isolates. Both techniques identified the maternal and infant isolates as the same strain, distinct from epidemiologically unrelated controls, thus confirming their common origin.
Vertical
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Citrobnctev koseri Transmission
REFERENCES Dan M, Sadan 0 (1989) VerticaI transmission of Citrobacter diversus from mother to infant. Ped Infect Dis J 8:63-64. Lin FC, Devoe WF, Morrison C, Libonati J, Powers I’, Gross RJ, Rowe 8, Israel E, Morris JG Jr. (1987) Outbreak of neonatal Citrobacfer diversus meningitis in a suburban hospital. Ped Infect Dis J 6:50-55. Finn A, Talbot GH, Anday E, Skros M, Provencher M, and Hoegg C (1988) Vertical transmission of Citrobacter diversus from mother to infant. Ped Infect Dis J 71293-294. Harvey BS, Koeuth T, Versalovic J, Woods CR, Lupski JR (1995) Vertical transmission of Citvobacter diversus documented by DNA fingerprinting. Infect Conf Hosp Epidemiol 16:564-569. Morris JG Jr, Feng-Ying CL, Morrison CB, Gross RJ, Khabbaz R, Maher KOD, Rowe B, Israel E, Libonati J (1986) Molecular epidemiology of neonatal meningitis due to Citrobacter diversus: A study of isolates from hospitals in Maryland. 1 Infect Dis 154:409414. Pfaller MA, Hollis RJ, Sader HS (1994) Chromosomal
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striction fragment analysis by pulsed-field gel electrophoresis. In Ed Clinical Microbiology Procedures Handbook (suppl 1) HD Isenberg. Washington, DC: American Society for Microbiology, p. 10.5C.12. Pfaller MA, Wendt C, Hollis RJ, Wenzel RI’, Fritschel SJ, Neubauer JJ, Herwaldt LA (1996) Comparative evaluation of an automated ribotyping system versus pulsedfield gel electrophoresis for epidemiological typing of clinical isolates of Escherichia coli and Pseudomonas aeruginosa from patients with recurrent Gram-negative bacteremia. Diagn Microbial Infect Dis submitted for publication. Walter E (1988) Vertical transmission of Citrobacter diversus from mother to infant. Ped Infect Dis J 7~675. Williams WW, Mariano J, Spurrier M, Donnell HD Jr, Breckenridge RL Jr, Anderson RL, Wachsmuth IK, Thornsberry C, Graham DR, Thibeault DW, Allen JR (1984) Nosocomial meningitis due to Citrobacter diversus in neonates; New aspects of the epidemiology. I Infect Dis 150~229-235.