Colonization and Persistence of Antibiotic-Resistant ...

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Apr 19, 2007 - Schrag, S. J., V. Perrot, and B. R. Levin. 1997. Adaptation to the fitness costs of antibiotic resistance in Escherichia coli. Proc. Biol. Sci.
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2008, p. 560–567 0095-1137/08/$08.00⫹0 doi:10.1128/JCM.00832-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 46, No. 2

Colonization and Persistence of Antibiotic-Resistant Enterobacteriaceae Strains in Infants Nursed in Two Neonatal Intensive Care Units in East London, United Kingdom䌤 Michael Millar,1* Alex Philpott,2 Mark Wilks,1 Angela Whiley,3 Simon Warwick,1 Enid Hennessy,3 Pietro Coen,4 Stephen Kempley,1 Fiona Stacey,3 and Kate Costeloe2 Barts and The London NHS Trust, Whitechapel, London E1 1BB, United Kingdom1; Homerton University Hospital NHS Foundation Trust, Homerton Row, London E9 6SR, United Kingdom2; Barts and The London NHS Trust, Queen Mary’s School of Medicine and Dentistry, Wolfson Institute, Charterhouse Square, London EC1M 6BQ, United Kingdom3; and University College Hospital, Euston Road, London, United Kingdom4 Received 19 April 2007/Returned for modification 1 October 2007/Accepted 13 November 2007

Stool samples were collected from infants nursed in two neonatal intensive care units (NICUs) in East London, United Kingdom. The aim of the study was to determine the incidence of and risk factors for the carriage of multiresistant Enterobacteriaceae strains (MRE; resistant to three or more classes of antibiotic) and the extent of the persistence of resistant strains following discharge. Sixty-two (50%) of 124 infants had acquired MRE by 2 weeks of postnatal age, and 69 (56%) infants had acquired MRE by discharge. The proportions of infants at 2 weeks carrying strains that were resistant to antibiotics were the following: tetracycline, 79%; amoxicillin, 78%; cephalosporins, 31%; trimethoprim, 20%; piperacillin-tazobactam, 11%; chloramphenicol, 9%; and aminoglycoside, 4%. A gestational age of less than 26 weeks was a risk factor for colonization with MRE at discharge, but not at 2 weeks. Analysis within a NICU showed that exposure of an infant to a specific antibiotic in the NICU was not a risk factor for the carriage of a strain resistant to that antibiotic. Estimates of persistence from discharge to 6 months were the following: for tetracycline, 57% (95% confidence intervals [CI], 0.35 to 0.87); chloramphenicol, 49% (95% CI, 0.20 to 0.83); trimethoprim, 45% (95% CI, 0.22 to 0.74); piperacillin-tazobactam, 42% (95% CI, 0.20 to 0.71); and augmentin, 34% (95% CI, 0.11 to 0.66). Strains resistant to cephalosporins or aminoglycosides showed lower levels of persistence. Nine of 34 infants (26.5%) with Escherichia coli and 4 (7.1%) of 56 infants with Klebsiella spp. at discharge carried strains indistinguishable by randomly amplified polymorphic DNA and antibiotic susceptibility patterns at 6 months. MRE were found at high frequency in the infants during their stay in the NICU and persisted in a proportion of infants.

creases the probability of MRE infection in vulnerable infants, with the potential for worse outcomes for infants associated with suboptimal empirical antibiotic treatment (21, 44). Many outbreaks of infection with antimicrobial-resistant Enterobacteriaceae strains have been described for infants nursed in neonatal intensive care units (NICUs) (4, 7, 11, 20). High rates of the colonization of infants in special-care baby units with MRE suggest that, in some units, MRE are endemic. There is evidence from previous studies, including a pilot study in a NICU in East London (31), that a large proportion of infants nursed in intensive care units acquire colonization with MRE, but there is little information on the duration of carriage in this population. Persistent colonization with resistant Enterobacteriaceae strains has potential implications not only for colonized infants but also for their contacts (12, 22). In this study, we have determined the frequency of and risk factors for the carriage of resistant strains by infants from two NICUs during the initial hospital stay and the persistence of resistant strains through infancy.

Gram-negative infection is a major cause of mortality in infants (38). A recent report from the Israeli Neonatal Network (25) estimates that sepsis due to Enterobacter spp., Escherichia spp., and Klebsiella spp. is associated with a 4.1-fold, 4.3-fold, and 6.3-fold risk of mortality, respectively, in the verylow-birth-weight population compared to that of sepsis associated with coagulase-negative staphylococci. Enterobacteriaceae strains resistant to three or more classes of antibiotic are endemic in many hospitals and account for a significant proportion of hospital-acquired bloodstream infections (8). Outbreaks of infection have been described for many different groups of patients (9, 10, 13, 24, 43), even extending to nonhospitalized individuals (5, 9, 15, 32). The choice of antibiotic for empirical therapy may be an important determinant of the clinical outcome for bloodstream infections caused by gram-negative bacteria (18, 42). An increasing prevalence of multiresistant Enterobacteriaceae strains (MRE; resistant to three or more classes of antibiotic) in-

* Corresponding author. Mailing address: Division of Infection, Barts and The London NHS Trust, 3rd Floor, Pathology and Pharmacy Building, 80 Newark St., London E1 2ES, United Kingdom. Phone: 20 3246 0296. Fax: 20 3246 0303. E-mail: michael.millar@bartsandthelondon .nhs.uk. 䌤 Published ahead of print on 26 November 2007.

MATERIALS AND METHODS Description of the two NICUs. Infants were recruited for a calendar year from July 2004 from two tertiary-level NICUs approximately 4 miles apart in East London, United Kingdom. Unit 1 (Barts and the London NHS Trust) has 23 medical cots (9 providing intensive care, 2 providing care for high-dependency

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infants, and 12 providing special care) and is colocated with a neonatal surgery unit. Unit 2 (Homerton University Hospital) has 33 cots (10 providing intensive care, 3 providing care for high-dependency infants, and 20 providing special care). The great majority of admissions are preterm infants requiring respiratory support. The local population is ethnically diverse; unit 1 has a large Bangladeshi population, and unit 2 has large African-Caribbean and Turkish populations. The units receive transfers from smaller neonatal units in the London area. The overall nursing staff/infant ratio in the units is 1:3. Infants with suspected early-onset sepsis are treated with penicillin and gentamicin in both units. Unit 1 uses piperacillin-tazobactam with vancomycin, and unit 2 uses flucloxacillin and gentamicin for suspected late-onset sepsis. Approval for this study was given by East London and the City Research Ethics Committee; all of the parents of participating infants gave informed consent. Inclusion criteria. All babies born during the study period and admitted to the unit within 24 h of birth and who remained for more than 14 days were eligible for recruitment into the study. Exclusion criteria. Babies with major congenital malformations and babies of non-English speakers for whom no advocate could be found were excluded. Those who died or who required gastrointestinal surgery before discharge from the hospital, those from whom samples were not obtained while in the hospital, and those from whom follow-up samples were unlikely to be obtained also were excluded. Sampling. Stool samples were collected at 2 weeks postnatal age and at the time of discharge. Parents also were approached to provide samples of stool from the infants at 6 and 12 months after discharge. Data collection. The initial clinical information included sex, gestational age, birth weight, ethnicity, maternal group B streptococcus (GBS) status (if known), the presence of a prolonged rupture of membranes (⬎24 h), the use of antibiotics in pregnancy and intrapartum, the use of antenatal corticosteroids, the number of births (i.e., single birth or multiple births), the mode of delivery, the use of mechanical ventilation, the use of a surfactant at birth, and the severity of early illness (clinical risk index for babies [CRIB] II score) (29). Additional data were collected each day and included the use and type of antibiotic, probable sepsis status (i.e., a positive culture from a normally sterile sample such as blood culture or cerebrospinal fluid), the type of feeds, the duration of ventilation via a tracheal tube, the use of a orogastric/nasogastric tube, the use of ranitidine, whether or not the infant had continued oxygen dependence at 28 weeks postnatal age and 36 weeks postmenstrual age, and the length of stay. At the time of collecting the 1-year-postdischarge stool sample, information about hospitalizations and antibiotic use during infancy was requested from the mother using a standardized questionnaire. Laboratory methods. Samples were processed in the routine microbiology laboratory at Barts and the London NHS Trust. Stool was added to a cryopreservative (brain heart infusion broth containing 10% glycerol, 0.0001% resazurin, and 0.02% dithiothreitol) and stored at ⫺70°C until processed. Samples were batch processed. Vials of frozen sample were thawed, and 20 ␮l of the fecal broth was subjected to 10-fold dilutions in phosphate-buffered saline. Fifty microliters of the 10⫺2, 10⫺4, and 10⫺6 dilutions was inoculated onto agar medium plates. Selective media for the growth of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci were included in the laboratory work. The agar plates were MacConkey agar (Oxoid Ltd., Basingstoke, United Kingdom), mannitol salt agar with oxacillin (Oxoid Ltd.), and Slanetz & Bartley agar with vancomycin (Oxoid Ltd.). Inoculated plates were incubated at 37°C for 24 to 36 h in air. Gram-negative bacilli that grew on MacConkey agar in air and were resistant to two or more antibiotics were identified. Representatives of different colony types were picked for identification and antibiotic susceptibility testing. Isolates were identified using standard laboratory methods, including the API 20E system (Biomerieux, Basingstoke, United Kingdom). Antibiotic susceptibility testing, including extended-spectrum ␤-lactam (ESBL) testing, was carried out using British Society for Antimicrobial Chemotherapy methods and interpretive criteria (www.BSAC.org.uk). The antibiotics tested were cefuroxime, ceftazidime, cefpodoxime, amoxicillin, gentamicin, piperacillin-tazobactam, augmentin, tetracycline, trimethoprim, amikacin, tobramycin, imipenem, ertapenem, tigecycline, colistin, and chloramphenicol. The presence of fully antibiotic-sensitive gram-negative bacilli was recorded, but these strains were not speciated. Cephalosporin resistance was defined as resistance to any of the cephalosporins tested; carbapenem resistance was defined as resistance to ertapenem or imipenem; and aminoglycoside resistance was defined as resistance to amikacin, gentamicin, or tobramycin. Typing of Escherichia coli and Klebsiella spp. DNA was extracted from discrete colonies growing on blood agar plates using the QIAamp mini kit (Qiagen Ltd., Crawley, United Kingdom) according to the manufacturer’s instructions. The

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DNA concentration of the extract was quantified by spectroscopy, and the concentration was adjusted to 2.5 ng ␮l⫺1. Randomly amplified polymorphic DNA (RAPD) patterns were generated with 5 ng of DNA from each isolate using ready-to-go RAPD analysis beads (Amersham Biosciences) according to the manufacturer’s instructions. Primer 2 (5⬘-G TTTCGCTCC) was used for E. coli, and primer 3 (5⬘-GTAGACCCGT) was used for Klebsiella spp. Products were analyzed on 2% agarose gels stained with ethidium bromide. Strains were described as indistinguishable if they were indistinguishable by both RAPD and antibiotic susceptibility patterns. Statistical methods. Multiresistance was defined as resistance to three or more classes of antibiotic. The classes were cephalosporins, amoxicillin, augmentin, tetracycline, trimethoprim, piperacillin-tazobactam, carbapenem, aminoglycoside, colistin, chloramphenicol, and ciprofloxacin. Logistic regression was used for the analyses using STATA 9; unless otherwise stated, results are from univariate analyses. For the separate classes of antibiotic resistance, the rates of acquisition and loss of a resistant strain between discharge and 6 months after discharge were assumed to be constant throughout the period for each class. The appendix gives further details of the methods used. The two groups, those resistant at discharge and those not, enabled separate differential equations to be given, which, when integrated, equated to the numbers of infants who had resistance at 6 months but did not have resistance at discharge and those who had no resistance at 6 months but did have resistance at discharge. These two equations could be solved (see the appendix) and rates of loss and acquisition estimated. Using these numbers, estimates were made for the proportion of children who had lost or gained resistant strains. The proportions with persisting resistance and their confidence intervals (CI) were calculated with the following formula: 1 ⫺ the proportion losing a strain. The CI for these values were calculated by bootstrapping the rates of acquisition and loss and using the confidence limits of the rates to estimate the CI of the proportions at 6 months. Because the formula for estimating acquisition and loss is undefined when one of the rates is very high or very low, the bootstrap method for CI excluded a significant proportion of samples in some cases; these are highlighted. The proportions of infants actually gaining (or losing) resistance to a class of antibiotic, over the 6 months must be lower than or equal to the proportion of infants who gained (or lost) resistance in this period. This is because some gainers could have then lost resistance and some losers could have gained resistance. If the lower binomial confidence limit from the actual proportions losing or gaining is larger than the bootstrap result or if the bootstrap result is unreliable, then the lower binomial has been substituted. This does not affect the lower CI of the proportion whose resistance persists. Exact CI for the binomial distributions have been used.

RESULTS There were 221 infants who fulfilled the inclusion criteria and were eligible for the study. Ninety-seven infants who fulfilled the inclusion criteria were not recruited to the study. These were infants predominantly from non-English-speaking parents for whom no advocate was found to provide informed consent. One hundred twenty-four infants were recruited (54 infants in unit 1 and 70 in unit 2). There were no significant differences in the baseline characteristics of patients admitted to the two units (Table 1). During the period of this study, there were no outbreaks of infection associated with specific strains of Enterobacteriaceae recorded or reported through routine infection control surveillance activities in either unit. In unit 1, there were nine patient episodes associated with the isolation of gram-negative bacilli from blood cultures during the period of this study. These episodes were caused by E. coli (n ⫽ 3), Klebsiella spp. (3), Serratia sp. (1), Enterobacter sp. (1), and Citrobacter sp. (1). All of the isolates were amoxicillin resistant. The Enterobacter spp. and Serratia spp. both were multiresistant. None of the Klebsiella spp. or E. coli isolates were ESBL producers, and none of them were gentamicin, piperacillin-tazobactam, or carbapenem resistant or multiresistant. In unit 2, there were eight patient episodes associated with the isolation of gram-negative

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J. CLIN. MICROBIOL. TABLE 1. Study population demographicsa Results for babies from:

Demographicb

No. of babies recruited Sex ratio (male/female) Median gestational age (wk) (IQR) Median birth wt (g) (IQR) Median birth wt SD (IQR) No. (%) of mothers with prolonged rupture of membranes (⬎24 h) No. (%) of mothers with GBS carriage on HVSc No. (%) of mothers receiving antibiotics during pregnancy No. (%) of mothers receiving intrapartum antibiotics No. (%) delivered by Caesarean section No. (%) receiving antenatal steroids No. (%) from multiple pregnancy Median CRIB II score (IQR) No. (%) receiving surfactant No. (%) receiving endotracheal ventilation Median duration of ventilation (IQR) No. (%) receiving oxygen at 28 days No. (%) receiving oxygen at 36 wk No. (%) receiving EBM at any time No. (%) receiving EBM at discharge No. (%) on oro/naso tube Median days on oro/naso tube (IQR) No. (%) receiving ranitidine Median no. of days in NICU (IQR) a b c

Both hospitals

Hospital 1

Hospital 2

124 71:53 (1.3:1) 31 (29–33) 1,530 (1,080–1,890) ⫺0.35 (⫺1.2–0.39) 33 (27)

54 (44) 28:26 (1:1) 31 (28–33) 1,521 (980–1,746) ⫺0.55 (⫺1.2–0.28) 12 (22)

70 (56) 43:27 (1.6:1) 31 (29–33) 1,545 (1,080–1,900) ⫺0.12 (⫺0.15–0.41) 21 (30)

7 (6)

4 (7)

3 (4)

15 (12)

7 (13)

8 (11)

19 (15)

6 (11)

13 (18)

78 (63) 100 (80) 27 (22) 4 (2–9) 55 (44) 60 (48) 0 (0–2) 29 (23) 14 (11) 112 (90) 70 (56) 119 (96) 25 (14–44) 9 (7) 32 (22–63)

36 (67) 38 (70) 8 (15) 4 (2–9) 20 (37) 25 (46) 0 (0–2) 13 (24) 4 (7) 47 (87) 30 (56) 51 (94) 27 (19–44) 5 (9) 33 (25–53)

42 (60) 62 (89) 19 (27) 4 (2–9) 35 (50) 35 (50) 0 (0–2) 16 (23) 10 (14) 65 (93) 40 (57) 68 (97) 26 (12–44) 4 (6) 31 (21–64)

Caucasian, 36%; West African, 20%; Bangladeshi, 16%; other, 28%. oro/naso tube, orogastric/nasogastric tube; EBM, expressed maternal breast milk; HVS, high vaginal swab. These numbers do not represent total rates of GBS carriage; only selected women were screened.

bacilli from blood cultures. These episodes were caused by Serratia sp. (n ⫽ 1), Klebsiella spp. (2), Pseudomonas aeruginosa (2), E. coli (1), Enterobacter sp. (1), and Acinetobacter sp. (1). The Serratia sp. was multiresistant. All of the isolates were amoxicillin resistant. None of the Klebsiella spp., Enterobacter spp., or E. coli isolates were ESBL producers, and none of them were gentamicin, piperacillin-tazobactam, or carbapenem resistant or multiresistant. Samples were received from 74 (60%) infants at 6 months after discharge, and 47 (38%) were received from infants at 12 months after discharge. Of the 124 infants recruited, 57 infants were discharged before 4 weeks, so that the 2-week sample also was the discharge sample. The proportion carrying strains resistant to each antibiotic at 2 weeks was 79% for tetracycline, 78% for amoxicillin, 31% for cephalosporins, 20% for trimethoprim, 11% for piperacillin-tazobactam, 9% for chloramphenicol, and 4% for aminoglycosides. The proportions of infants colonized with Enterobacteriaceae strains resistant to each antibiotic class at 2 weeks, at discharge, and at 6 and 12 months after discharge are shown in Fig. 1. One strain resistant to tigecycline was detected in the 2-week samples, and no strain was resistant to carbapenems. The majority of infants carrying cephalosporin-resistant strains were carrying cephalosporin-resistant Enterobacter spp. (24 of 38 [63.2%] infants at discharge). Three infants carried ESBL-producing strains at discharge, and four infants carried ESBL-producing strains at 2 weeks. The proportions of infant samples from which MRE were

isolated at the various time points were the following: at 2 weeks, 50%; at discharge, 56%; at 6 months, 42%; and at 1 year, 51%. At discharge, 34 (27%) of 124 infants carried E. coli strains resistant to two or more antibiotics (of which more than 80% were MRE), 56 (45.2%) carried Klebsiella spp. (of which 41% were MRE), 24 (19.4%) carried Enterobacter spp., and 23 (18.5%) carried other Enterobacteriaceae species. No vancomycin-resistant enterococci were isolated in the discharge samples. A methicillin-resistant S. aureus strain was isolated from samples from one infant at 2 weeks postnatal age and at discharge. Questionnaires were received for 29 (62%) of the infants from whom stool samples were received at 1 year after discharge. Nine of those 29 infants had received antibiotics, and 6 had been hospitalized in the year following discharge. There were no statistically significant univariate associations of predetermined risk factors with the carriage of MRE at 2 weeks (Table 2), nor was there any suggestion that low-gestational-age infants were more at risk of colonization with MRE in the first 2 weeks. However, being born at ⬍26 weeks of gestational age was significantly associated with more colonization by MRE at discharge, and in those babies born after 26 weeks the use of a cephalosporin was associated with less MRE colonization at discharge. The antibiotic exposure of an individual was not significantly positively associated with resistance to that antibiotic, except for piperacillin-tazobactam. Thirty-eight percent of those exposed to piperacillin-tazobactam became colonized with a re-

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FIG. 1. Proportions of infant samples with Enterobacteriaceae strains resistant to each antibiotic at 2 weeks, at discharge, and at 6 and 12 months after discharge. Antibiotics: 1, amoxicillin; 2, augmentin; 3, trimethoprim; 4, tetracycline; 5, chloramphenicol; 6, colistin; 7, aminoglycoside (gentamicin, tobramycin, or amikacin); 8, cephalosporin (cefuroxime, ceftazidime, or cefpodoxime); 9, carbapenem (imipenem or ertapenem); and 10, piperacillin-tazobactam; 11, ciprofloxacin.

sistant strain by discharge, whereas 15% of those not exposed to piperacillin-tazobactam became colonized with a resistant strain by discharge (P ⫽ 0.054; Fisher’s exact test). However, more infants not exposed to piperacillin-tazobactam were colonized with piperacillin-tazobactam-resistant strains in the unit in which piperacillin-tazobactam was part of the protocol (20%) than in the unit in which piperacillin-tazobactam was not used (13%). If only data for infants from the center using the antibiotic were analyzed, the association was not significant. The odds ratio for resistance if an infant was administered piperacillin-tazobactam was 2.6 (95% CI, 0.66 to 10). An investigation of the number of classes of resistance at 2 weeks and at discharge showed that for those colonized with a strain resistant to any antibiotic at 2 weeks, birth at ⬍26 weeks of gestational age was associated with more classes of resistance at discharge (2.4 more classes; 95% CI, 0.7 to 4.0; P ⫽ 0.006); however, when adjusted for gestational age, the length of stay was not associated with more classes of resistance at discharge. However, for those without a resistant strain at 2 weeks, the length of stay was independently associated with gaining classes of resistance, with approximately one extra class of resistance per 4 weeks of stay (95% CI, 0.21 to 1.9; P ⫽ 0.017). Persistence of resistant gram-negative bacilli following discharge from the NICU. There was a decline in the proportion of infants carrying strains resistant to cephalosporins, piperacillin-tazobactam, or aminoglycosides following discharge, but not of those carrying strains resistant to amoxicillin, trimethoprim, colistin, tetracycline, or chloramphenicol. In order to demonstrate the persistence of specific strains, E. coli and Klebsiella spp. were typed by RAPD and antibiotic

susceptibility patterns. E. coli was isolated from 34 infants (27.4%) at discharge, from 45 (60.8%) infants at 6 months, and from 27 (57.4%) at 1 year. Strains of E. coli from the 12 infants who were colonized both at discharge and at 6 months were typed. Pairs of E. coli isolates from 9 (75%) of these 12 infants gave indistinguishable RAPD and antibiotic susceptibility patterns, showing that 9 (26.5%; 95% CI, 12.9 to 44%) of 34 infants with E. coli at discharge carried an indistinguishable strain 6 months later. Seven of the 12 infants lost a strain of E. coli, and four gained a new strain. E. coli was isolated from 27 (57.4%) infants at 12 months. Nineteen pairs of isolates from infants colonized at both 6 and 12 months were typed. Eight (42%) of the 19 pairs of isolates were indistinguishable by RAPD and susceptibility patterns, showing that 8 (17.8%; 95% CI, 8 to 32%) of 45 infants carried an indistinguishable strain at 6 and 12 months. Four (11.8%; 95% CI, 3.3 to 27%) of 34 infants who carried E. coli at discharge carried an indistinguishable E. coli isolate at 1 year. Klebsiella spp. were isolated from 56 (45.2%) infants at discharge, 27 (36.5%) infants at 6 months, and 17 infants (36.2%) at 12 months. Discharge and 6-month pairs of isolates were typed from 16 infants. Of these pairs, four (25%) were indistinguishable by RAPD and antibiotic susceptibility patterns, showing that four (7.1%; 95% CI, 2.0 to 17%) carried indistinguishable strains at discharge and at 6 months. Twelve infants lost a strain and 12 gained a strain between discharge and 6 months. The proportions of infants carrying an indistinguishable strain of E. coli at discharge and 6 months later was significantly higher than those for Klebsiella spp. (26.5% for E. coli versus 7.1% for Klebsiella spp.; chi-square test, 6.2; P ⬍ 0.025).

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TABLE 2. Risk factors for colonization with MRE at 2 weeks and at discharge from univariate logistic regression Odds ratio (95% CI) for:

2 wk

Discharge

Infants born at ⱖ26 wk gestational age (at discharge)

Male sex Multiple birth Mode of delivery (LSCS) Gestational age (per wk) Gestational age of ⬍26 wk Birth wt SD score, per SD

1.22 (0.60, 2.49) 1.96 (0.82, 4.72) 1.00 (0.48, 2.07) 1.04 (0.94, 1.15) 0.79 (0.20, 3.08) 0.79 (0.58, 1.08)

1.83 (0.89, 3.76) 0.56 (0.24, 1.33) 0.95 (0.45, 1.97) 0.93 (0.84, 1.02) —a 1.03 (0.76, 1.40)

1.67 (0.79, 3.50) 0.67 (0.28, 1.59) 1.23 (0.57, 2.67) 0.99 (0.89, 1.11) NA 0.99 (0.72, 1.36)

Antenatal factors Antepartum antibiotics Prolonged rupture of membranes (⬎24 h) Antenatal steroids; any vs none

0.86 (0.29, 2.53) 0.56 (0.25, 1.25) 0.53 (0.21, 1.33)

0.90 (0.30, 2.65) 0.67 (0.30, 1.50) 0.87 (0.35, 2.15)

0.76 (0.24, 2.42) 0.56 (0.24, 1.31) 0.80 (0.32, 2.02)

Early status in hospital CRIB II score, per point Surfactant

0.99 (0.91, 1.08) 1.58 (0.78, 3.23)

1.12 (1.02, 1.22) 1.80 (0.87, 3.72)

1.05 (0.95, 1.17) 1.34 (0.63, 2.83)

Ventilation requirements ETT ventilation ETT ventilation for more than 3 days Oxygen at 28 days Oxygen at 36 wk

1.79 (0.88, 3.66) 0.59 (0.21, 1.64) 1.09 (0.48, 2.51) 1.38 (0.45, 4.25)

1.95 (0.95, 4.00) 3.25 (1.00, 10.51) 3.21 (1.25, 8.22) 3.29 (0.87, 12.43)

1.50 (0.72, 3.14) 1.42 (0.38, 5.31) 1.90 (0.70, 5.17) 2.67 (0.67, 10.62)

Feeding Orogastric/nasogastric tube feeding Any maternal breast milk in hospital Ranitidine use

0.66 (0.11, 4.07) Not known 0.79 (0.20, 3.08)

0.30 (0.03, 2.77) 0.60 (0.17, 2.10) 2.99 (0.60, 15.02)

0.26 (0.03, 2.39) 0.51 (0.14, 1.80) 2.41 (0.45, 12.96)

Antibiotic use in hospital At any time prior to sample After 48 h Cephalosporin use prior to sample Use of second line of antibioticsc Flucloxacillin use

0.82 (0.24, 2.8) 0.72 (0.35, 1.47) —b 1.00 (0.43, 2.3) 1.41 (0.55, 3.6)

0.89 (0.27, 2.96) 1.22 (0.60, 2.51) 0.19 (0.02, 1.73) 3.13 (1.28, 7.70)d 2.9 (1.22, 6.90)d

0.76 (0.23, 2.54) 0.87 (0.41, 1.84) —f 2.14 (0.83, 5.49) 1.86 (0.74, 4.64)

Other hospital data Length of stay, per wk

0.98 (0.91, 1.06)

1.09 (1.00, 1.19)

1.03 (0.93, 1.13)

Risk factor

e

All babies at:

All babies with a gestational age of ⬍26 weeks were colonized with MRE at discharge; P ⫽ 0.004 (Fisher’s exact test). NA, not applicable. No babies given cephalosporin prior to the 2-week sample were colonized with MRE at 2 weeks (P ⫽ 0.50). New prescriptions were given after day 5 of life. d 0.05 ⬎ P ⬎ 0.01. e LSCS, lower-segment Caesarean section; ETT, endotracheal tube. f No babies with a gestational age of 26 weeks or more who were given cephalosporin prior to discharge were colonized with MRE at discharge (P ⫽ 0.049). a b c

Estimates of persistence of resistance. Estimates of the extent of persistence were modeled (Table 3) and ranged from 0 to 57% depending on the antibiotic. The estimated levels of persistence were more than 20% for the majority of the antibiotic classes. The estimated persistence rates for augmentin, trimethoprim, tetracycline, chloramphenicol, and piperacillintazobactam were high (⬎33%) and excluded the persistence of less than 20% for trimethoprim, chloramphenicol, and piperacillin-tazobactam. The lower CI may be unreliable for antibiotics for which more than 10% of the bootstrap samples did not give an estimate, but for the remainder, for which more than 99% of samples gave estimates, they should be reliable. We have 95% confidence that trimethoprim and chloramphenicol resistance will persist in at least 20% of similar children. Because the acquisition of piperacillin-tazobactam is low and because it is unlikely that more than 1 of the 14 infants with resistance lost and reacquired resistance, we can assume that in 7/14 the

resistance really persisted; therefore, the 95% CI for persistence would be 0.23 to 0.77, so persistence is likely for at least 20% of infants during a 6-month period. DISCUSSION This study confirms that a large proportion of infants nursed in two geographically separated NICUs became colonized with MRE, including strains that are resistant to antibiotics (such as chloramphenicol and tetracyclines) that are rarely administered systemically to infants. This study also shows that the bowel flora is a major reservoir for MRE in infants nursed in NICUs, and that the acquisition of resistant strains occurs by 2 weeks of postnatal age. There are no previous reports describing the persistence of antimicrobial-resistant gram-negative bacteria in infants following discharge from a NICU. This study suggests that there are high levels of persistence of resistant Enterobacteriaceae strains in this population.

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TABLE 3. Numbers of infants acquiring, losing, or retaining strains resistant to each antibiotic between discharge and 6 months after discharge

Antibiotic

a

Cephalosporin* Amoxicillin* Augmentin Trimethoprim Tetracycline* Gentamicin* Amikacin Tobramycin Ciprofloxacin* Imipenem Chloramphenicol Piperacillin-tazobactam* Colistin* a b c

No. of infants losing or gaining strain/total no. of infants

Estimated rate of acquisition or loss after 6 mo

Estimated proportion (odds ratio 关95% CI兴) gaining resistance within 6 mo of discharge or with persisting resistance up to 6 mo

Lost

Gained

Acquisition

Loss

Gaining resistanceb

Persisting resistancec

16/22 16/57 12/21 11/23 14/62 5/6 2/2 5/5 3/4 2/2 5/11 8/14 8/10

9/52 12/17 10/53 10/51 8/12 3/68 1/71 2/68 3/70 0/72 10/63 1/60 6/64

0.44 3.08 0.35 0.33 1.67 0.11 ND ND 0.09 ND 0.25 0.025 0.24

1.86 1.23 1.07 0.80 0.56 1.99 ND ND 1.49 ND 0.70 0.86 2.00

0.36 (0.08,0.59) 0.95 (0.44,1.0) 0.30 (0.09,0.51) 0.28 (0.10,0.47) 0.81 (0.35,0.96) 0.10 (0.01,0.20) ND (0, ND) ND (0, ND) 0.08 (0.01,0.16) ND (0, ND) 0.22 (0.08,0.16) 0.02 (0,0.08) 0.21 (0.04,0.38)

0.16 (0.03,0.50) 0.29 (0.09,0.83) 0.34 (0.11,0.66) 0.45 (0.22,0.74) 0.57 (0.35,0.87) 0.14 (0.04,0.64) ND (ND, 0.85) ND (ND, 0.52) 0.23 (0.08,0.81) ND (0, 0.85) 0.49 (0.20,0.83) 0.42 (0.20,0.71) 0.13 (0.02,0.56)

An asterisk indicates that more than 10% of bootstrap samples for this antibiotic were not able to be calculated. ND, not done. The lower confidence limit was calculated from the binomial CI of the proportion gaining resistance (derived from the Gained column). The upper confidence limit was calculated from the binomial CI of the proportion losing resistance (derived from the Lost column).

There is an increasing trend toward the use of intrapartum antibiotics for the prevention of GBS disease (35) and to improve the pregnancy outcome in some groups, such as those with preterm rupture of membranes. There are conflicting reports as to whether this has resulted in an increase in the absolute numbers of episodes of early-onset sepsis caused by gram-negative bacteria (25, 33, 37), but there is evidence to suggest that antimicrobial resistance is increasing in those gram-negative isolates associated with both early- and lateonset neonatal sepsis (17, 35, 37). In a report from Washington, DC, 17% of infants admitted to a NICU became colonized with antibiotic-resistant Enterobacteriaceae strains, and 14% of colonized infants developed infection (36). A temporal relationship of colonization preceding bloodstream infection with Enterobacteriaceae strains also has been described (6). In this study, the colonization of infants early in life with MRE was frequent, but despite this there were few episodes of bacteremia associated with MRE (during the inpatient stay). In a recent study from the United States, 10% of infants presenting to a children’s hospital with fever between 57 and 180 days of age were diagnosed with serious bacterial sepsis, of which the great majority were urinary tract infections caused by gram-negative bacilli (most frequently E. coli) (16). In this study, we did not collect detailed information on postdischarge events, although 6 (21%) of 29 infants were reported by caretakers to have been admitted to the hospital in the 12 months following discharge. Antibiotic policies for the treatment of late-onset sepsis in neonates generally recommend an aminoglycoside or a cephalosporin (see, for example, reference 26). These also are mainstays for the treatment of suspected sepsis during the first year of life. In this study, the proportion of infants carrying strains resistant to cephalosporins or aminoglycosides declined after discharge, and cephalosporin- and aminoglycoside-resistant strains were among the least likely to persist. The small proportions carrying ESBL-producing strains may have been an important factor in determining the relatively low level of persistence of cephalosporin resistance in the study population. In this study, strains resistant to two or more antibiotics were

identified using the API system. E. coli and Klebsiella spp. were typed by RAPD. Indistinguishable strains of E. coli were isolated from 26.5% of infants at discharge and at 6 months. High levels of persistence of E. coli in infants have been reported from another northern European country (Sweden) (27), with 53% of infants carrying only one strain of E. coli in the first 6 months of life. The same group (19, 28) reported the persistence of tetracycline-resistant E. coli in Swedish infants, and they also reported that tetracycline-resistant strains were more likely to carry colonization factors. In this study, estimates of the persistence of Enterobacteriaceae strains were highest for tetracycline- and chloramphenicol-resistant strains. Amoxicillin and related antibiotics frequently are used in infants, but infants are rarely systematically exposed to chloramphenicol or tetracyclines. Chloramphenicol-resistant E. coli strains are widespread in farm animals (3), and this may reflect the association of chloramphenicol resistance with colonization factors (41) and/or linkages with other resistance determinants (39). Compensatory mechanisms that preclude a reversion to antibiotic sensitivity (2, 34) also may be important determinants of persistent resistance to specific antibiotics in populations for which those specific antibiotics are infrequently used. Many previous studies of antibiotic-resistant gram-negative bacilli have taken place during outbreaks involving single bacterial strains or species in single NICUs and have used a case control design. For example, Linkin et al. reported (23) that, during an outbreak, the risk factors for colonization or infection were low gestational age and exposure to expanded-spectrum cephalosporins. In a study of children during a period of increased isolation of resistant gram-negative bacteria, the administration of an extended-spectrum cephalosporin was reported as a risk factor for infection with a resistant gram-negative bacilli (44). Risk factor analyses for single strains or species may not be generally applicable to antibiotic-resistant Enterobacteriaceae strains, and risk factors for infection may be different from risk factors for colonization. Episodes of colonization and infection frequently are accumulated in analyses. Case control studies may not accurately estimate the extent to which antibiotics are risk factors for the

566

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J. CLIN. MICROBIOL.

acquisition of resistant strains (14, 30). In particular, case control studies may overemphasize antibiotic exposure as a risk factor for the carriage of resistant strains. The study reported here was a cohort study carried out during a non-outbreak period (as attested to by the low frequency of isolation of MRE from blood cultures during the time period of this study), and we did not find that antibiotic exposure was a risk factor for colonization with resistant Enterobacteriaceae strains in individual infants (apart from piperacillin-tazobactam) when the results from the two units were aggregated. When looked at on the level of individual units, exposure to piperacillin-tazobactam was not a risk factor for a piperacillin-tazobactam-resistant strain. These findings do not preclude the possibility that exposure to specific antibiotics is a risk for colonization with strains resistant to those antibiotics, because high levels of cross-infection may have obscured the effects of antibiotic exposure. We did find that low gestational age (⬍26 weeks) was a risk factor for the carriage of antibiotic-resistant Enterobacteriaceae strains at discharge, although it was not a risk factor for acquisition by 2 weeks. Previous studies have reported early colonization and high levels of cross-colonization with antibioticresistant gram-negative rods in infants nursed in intensive care units (1, 40). The risk factor findings of this study could be explained if colonization with resistant Enterobacteriaceae strains was a function of the intensity of caretaker-infant interactions and the length of time spent on the unit, with similar levels of intensity of care for infants of various gestational ages in the first 2 weeks following admission. MRE were found at high frequency in the infants during their stay in the NICUs. Gestational age and antibiotic exposure were not risk factors for the colonization of individual infants with MRE at 2 weeks of age. Antibiotic-resistant strains persisted in a portion of infants. The clinical and public health implications of the carriage and the persistence of carriage of MRE in this population of infants remain to be investigated. APPENDIX Methods for the determination of persistence of resistant Enterobacteriaceae strains. (i) Assumptions. The following assumptions were made: the rate of acquisition is constant; p (for “plus”) is the rate of strain acquisition during a fixed time period; the rate of the loss of resistance is constant; m (for “minus”) is the rate of dropping an acquired strain during a fixed time period; the time period is from 0 to 1. There are two scenarios providing data. e is the experimental constant, and c is the constant for integration. (a) Scenario A. In the first scenario, those infants without resistance at the start of the follow-up period give numbers with resistance at the end of the period. Let N(t) be the proportion of infants without resistance at time t. A is the proportion of infants who gained resistance by time 1. (b) Scenario B. In the second scenario, those infants with resistance at the start of the follow-up period give numbers without resistance at the end of the period. Let R(t) be the proportion with resistance (R) at time t. L is the proportion of infants who lost resistance by time 1. Equations. The following differential (d) equation is for scenario A: dN ⫽ dt[⫺pN ⫹ m(1 ⫺ N)] (the proportion of infants acquiring resistance reduces the size of the proportion of in-

fants without resistance, and the proportion losing resistance increases the number of infants without resistance); therefore, dN/dt ⫽ m ⫺ (p ⫹ m)N. Similarly, for scenario B, dR ⫽ dt[⫺mR ⫹ p(1 ⫺ R)] (the number of infants losing resistance reduces the number of infants with resistance, and the number gaining resistance increases the number with resistance); therefore, dR/dt ⫽ p ⫺ (p ⫹ m)R. Applying the integrating factor (IF) method to scenario A yields dN/dt ⫽ m ⫺ (p ⫹ m)N, which can be rearranged to dN/dt ⫹ (p ⫹ m)N ⫽ m. IF ⫽ exp[兰(p ⫹ m)dt] ⫽ e(p ⫹ m)t (where exp refers to the exponential function). c is a constant. N共t兲 ⫽ e⫺共 p ⫹ m兲t

再冕

关m ⫻ e共 p ⫹ m兲t兴dt



⫽ 关e⫺共 p ⫹ m兲t兴关m/共m ⫹ p兲兴关e共 p ⫹ m兲t ⫹ c兴 ⫽ 关m/共m ⫹ p兲兴兵1 ⫹ 关c ⫻ e⫺共 p ⫹ m兲t兴其 When t ⫽ 0, N ⫽ 1 ⫽ [m/(m ⫹ p)](1 ⫹ c), hence c ⫽ p/m, resulting in N(t) ⫽ 1/(m ⫹ p){m ⫹ [p ⫺ e⫺(p ⫹ m)t]}. Applying the IF method to scenario B yields dR/dt ⫽ p ⫺ (p ⫹ m)R, which can be rearranged to dR ⫹ (p ⫹ m)R ⫽ p. For the following equation, IF ⫽ exp[兰(p ⫹ m)dt ⫽ e(p ⫹ m)t] and 1/IF ⫽ e⫺(p ⫹ m)t. R共t兲 ⫽ 关e⫺共 p ⫹ m兲t兴

再冕

关 p ⫻ e共 p ⫹ m兲t兴dt



⫽ 关e⫺共 p ⫹ m兲t兴关 p/共 p ⫹ m兲兴关e共 p ⫹ m兲t ⫹ c兴 ⫽ 关 p共 p ⫹ m兲兴兵1 ⫹ c关e⫺共 p ⫹ m兲t兴其 When t ⫽ 0 and R ⫽ 1, c ⫽ m/p. Hence, R(t) ⫽ [1/(p ⫹ m)] {p ⫹ m[e⫺(p ⫹ m)t]}. When t ⫽ 1, R ⫽ 1 ⫺ L, and N ⫽ 1 ⫺ A, 共m ⫹ p兲 ⫺ L共 p ⫹ m兲 ⫽ p ⫹ m关e⫺共 p ⫹ m兲兴 L共 p ⫹ m兲 ⫽ m关1 ⫺ e⫺共 p ⫹ m兲兴

(1)

共1 ⫺ A兲共 p ⫹ m兲 ⫽ m ⫹ p关e⫺共 p ⫹ m兲兴 共 p ⫹ m兲 ⫺ A共 p ⫹ m兲 ⫽ m ⫹ p关e⫺共 p ⫹ m兲兴 A共 p ⫹ m兲 ⫽ p关1 ⫺ e⫺共 p ⫹ m兲兴

(2)

If equation 1 is divided by equation 2, then L/A ⫽ m/p and m ⫽ p(L/A). For the following substitution for equation 1, let L/A ⫽ q and m ⫽ qp. L关共1 ⫹ q兲p兴 ⫽ p兵q关1 ⫺ e⫺共1 ⫹ q兲p兴其 L共1 ⫹ q兲 ⫽ q ⫺ q关e⫺共1 ⫹ q兲p兴 ⫺ 共L ⫹ Lq ⫺ q兲/q ⫽ e⫺共1 ⫹ q兲p p ⫽ ⫺ 1/共1 ⫹ q兲兵loge关 ⫺ 共L ⫹ Lq ⫺ q兲/q兴其 q and L are known; hence, p and m can be calculated.

ACKNOWLEDGMENT This study was funded by a research grant from the Research Advisory Board, Barts and The London Charitable Trustees.

VOL. 46, 2008

ANTIBIOTIC-RESISTANT ENTEROBACTERIACEAE IN INFANTS REFERENCES

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