Epidemiological analysis of strains of methicillin-resistant Staphylococcus aureus (MRSA) infection in the nursery; prognosis of MRSA carrier infants

rournal

qf Hospital

Injection

(1995)

31, 123-134

Epidemiological analysis of strains of methicillinresistant Staphylococcus aureus (MRSA) infection in the nursery; prognosis of MRSA carrier infants T. Mitsuda,”

K. Arai,?

S. Fujita*

and S. Yokota*

Yokohama City University School of Medicine, “Department of Pediatrics, 3-9 Fukuura, Kanazawa-Ku, Yokohama, 236 and TDepartment of Microbiology, Yokohama City Institute of Health, 1-2-l 7 Takigashira, Isogo-Ku, Yokohama, 235, Japan Accepted for publication

20 &ne

1995

Summary: Forty-five neonates who carried methicillin-resistant StaphJ,lococcus aweus (MRSA) were studied. Retrospective molecular analysis using pulsed-field gel electrophoresis showed three separate MRSA epidemics in the nursery. Strains of MRSA isolated from the neonates were also isolated from the hospital environment and health care providers. Clinical manifestations included skin pustules (eight patients), conjunctivitis (four patients), or other minor infections (two patients). No neonate developed systemic infection. The prevalence of MRSA decreased with age. At one year, three (14.3%) of 21 infants that had carried MRSA at six days remained carriers and only two (1.1%) of 180 infants in a control ‘S. aureus-negative at six days’ group carried IL’IRSA. Keywords: pulsed-field

Methicillin-resistant gel electrophoresis.

Staphylococcus

auyeus;

neonatal

infection;

Introduction

Staphylococcus auyeus exists in the bacterial flora of 2535% of the population. This organism can cause severe infections in mothers and infants in the perinatal period.’ The prevalence of methicillin-resistant S. aureus (MRSA) has increased in clinical samples since the 197Os.*,” In Japan, 60-90% of S. aureus isolates from inpatients were reported recently to be methicillin-resistant. Patients with immune disturbances, including premature and newborn infants, are highly susceptible to infection by this organism.4’5 Epidemics of S. aureus in the nursery or neonatal intensive care unit are becoming a serious problem.G8 We investigated the epidemiology of MRSA infection in infants in the nursery of a maternity hospital using pulsed-field gel electrophoresis (PFGE) during three epidemics, and conducted a follow-up study of MRSA carriage among these infants. correspondence

to: T. Mitsuda

124

T. Mitsuda Subjects

and

et al. methods

Subjects This study was conducted between August 1991 and July 1993 at Yokohama Municipal Maternity Hospital, during which time we observed three outbreaks of MRSA infection in the nursery. Around 800 infants are delivered annually. Care is provided by more than 50 staff members including 46 nurses and six doctors. Prior to delivery, the perineum is routinely disinfected using a 0.025% ‘Hiamine’ solution containing benzethonium chloride, but the vagina is not disinfected. Neonates are admitted to the nursery. Mothers and their infants stay in the rooming-in unit from 24 h after birth. In the present study, we obtained nasal swabs from 442 infants at six days, and tested them for S. Lzureus. Infants found to carry MRSA, and those who were negative for any strain of S. aureus (controls), were studied. The infants who carried MRSA at six days were also examined at 1, 3, 6 and 12 months, while the controls were examined at 1, 3 and 12 months. S. aureus isolates were classified into four groups: MRSA-positive; methicillinsusceptible (MSSA)-positive; MRSA and MSSA-positive; and S. aureusnegative (controls). The number of infants evaluated at follow-up differed from those initially studied, as they were examined only when brought voluntarily to the well-baby clinic by their parents. To evaluate the problem of MRSA in the environment, specimens were collected from 24 different sites of the hospital during the three epidemics. They were taken by swabbing the floor of the nursery and the maternity wards from areas that drainage areas of the nursery bathtubs. measured 10 x 10 cm*, including Specimens were collected monthly for two years. Nasal swabs were obtained from the health care providers, including all the nurses and doctors in October 1991, January 1992 and June 1992. Bacterial isolates and biological characterization of isolated MRSA Samples were collected with sterilized swabs that were immediately streaked onto salt egg yolk agar medium using commercially available S. aweusselective agar base (Nissui Pharmaceutical Co., Tokyo, Japan) and incubated at 35°C for 24 h. Isolates were identified as S. aweus by their ability to ferment mannitol, a positive egg yolk reaction,’ and a positive coagulase test. Confirmation was obtained by using the rapid latex test for the identification of S. aureus (Staphaurex, Murex Diagnostics Ltd, Dartford, UK). Five colonies were randomly selected from each plate for further analysis. Methicillin resistance was determined from minimal inhibitory concentration (MIC) values obtained by the macrodilution broth method with 24 h incubation at 35°C performed according to the method of the National Committee of Clinical Laboratory Standards (MIC of methicillin for MRSA 2 16 pg/mL).” Coagulase-typing, enterotoxin-typing and tests for toxic shock syndrome

Epidemiological

toxin-I Dennka

analysis

of MRSA

infection

(TSST-I) production were performed Seiken (Tokyo, Japan).

in the nursery

using

kits obtained

125

from

Chromosomal DNA analysis by PFGE Chromosomal DNA was prepared according to the protocol of Smith and Cantor with minor modifications.““* Five hundred microlitres of an overnight culture in heart-infusion broth was harvested and resuspended in 150 FL. of Pett IV solution [I M NaCl, IO mM EDTA (pH 8.0)]. This suspens.ion was mixed with an equal volume of 2% low-meltingtemperature agarose (‘InCert’ agarose, FMC Co., Rockland, ME, USA) and allowed to solidify in a mould (250 pL). The agarose block was incubated overnight at 37°C in a lysis solution [I M NaCl, 0.1 M EDTA (pH S.O), IO mM Tris-HCl (pH S.O), 0.5% (w/v) Brij 58, 0.2% (w/v) deoxycholate, 0.5% (w/v) sarkosyl] supplemented with lysozyme [5 mg/ mL (Wako Pure Chemical Co., Tokyo, Japan)] and lysostaphin [IO0 U/ mL (Sigma Chemical Co.)]. After incubation overnight at 37”C, the block was immersed in I mL of proteolysis buffer [0.25 &I EDTA (pH S.O), I”/0 sarkosyl (w/v), 0.1 mg/mL proteinase K (Boeringer-Mannheim)], and digested for 24 h at 52°C. The plugs were rinsed three times in I mM phenylmethylsulphonyl fluoride (Sigma Chemical Co.) in TE buffer [IO mM Tris-HCl (pH 8*0), I mM EDTA (pH 8.0) for IO min at 4°C. The plugs were equilibrated in TE buffer by washing three times for IO min at 4°C. Twenty microlitres of thinly sliced sections from an agarose block were digested with 20 U of SmaI (New England Biolabs, Inc., Beverly, MA, USA) f or 6 h at 30°C and then electrophoresed through a 0.9% agarose gel (‘SepRate’-PFGE, Amersham, Bucks, UK) in 0.5 X TBE buffer [IX TBE buffer: 0.1 M Tris-HCl, 0.1 M boric acid, 2 rnlv EDTA (pH S.O)] at 14°C using a contour-clamped homogeneous electric field (CHEF) system (‘CHEF-DR’ II: BioRad Lab., Richmond, CA, USA). Lambda ladder DNA concatemers (BioRad) were used as molecular weight markers. Electrophoresis was performed at 17OV for 22 h, with pulse times from IO-100 s. Gels were stained with ethidium bromide (EtBr) for 25 min, washed with distilled w:ater, then photographed under U.V. radiation (302 nm). Statistical methods The x2 test was used to evaluate as statistically significant.

the data. A level of PcO.05

was accepted

Results

Isolation of MRSA; prognosis of MRSA carriers and of S. aureus-negative (control) infants Of the 442 neonates tested, 45 (10.2(Y)I) were positive for MRSA, 30 (6.8%) were positive for MSSA and 367 (83.0%) were free of S. aweus. Four

(kb)

Figure 1. Pulsed-field gel electrophoresis analysis of 45 methicillin-resistant StuphyZococcus aureus (MRSA) strains isolated from 6-day-old infants. Date of isolation appears at the top of the figure. Numbers l-45 represent the MRSA strains isolated from the 45 infants. Lambda (h) at both ends and in the middle represents the molecular weight markers (lambda ladder DNA concatemers).

200-

400-

600-

-+

I.

group

Prognosis

neonates*

neonates* 6 days? 1 month+ 3 months 6 months? 12 months 6 days 1 month? 3 months? 12 months

he

45 27 22 31 21 367 360 269 180

neonates

1’8. S.

aurrus-negative

neonates

(P=O.O34).

(8.9%) (18.5%) (40.9%) (25.8%) (28.6%)

they were examined

(MSSA)

carrier

i?

in the MRSA-positive

4

i

z

5’

6’ 3

E $ 5. ;i: 2

%

g

3w. O_ UQ ij* E. 92 $

in the iXIRSA-positive

only,

(0%) (14.8%) (22.7%) (58.1%) (57.1%) (100%) (61.7%) (45.7%) (71.1%) and MSSA

f: 5 18 12 367 222 123 128

S. aureus negative no. (%)

for SIRSA

12: (33(:;; 132 (49-l %) SO (27.8%)

4 5 9 8 6

YISS.4 positive no. (X,)

S. aureus

one month and t\co at six months group. positivity was significantly higher

because

_______ 45 (100%) 19 (70.4%) 8 (36.4%) 7 (22.6%) 3 (14.3%)$ 0 (0%) 20 (5.6%) 1.5 (5.6%) 2 (l.l’/o)$

MRSA positive no. (%)

aureus IIWRSA] and nlethicillin-susceptible neonates and S. aureus-negative neonates

No. of infants examined

Staphylococcus state in r%fRS,4-positice

of ,net/lic,illilz-rrsistalrt

* The number of infants examined at follow-up differed from that admitted to stud), \\hen brought to the L\ell-baby chnic hy their parents. t LIRS.4 and hlSS.4 \\ere isolated from some infants: four infants at six days. one at group; three at one month and onr at three months in the S. aureus-negative neonate $ Follow-up study at 12 months in both groups showed that the prevalence of blRSA

S. nzrrezcs-negative

hIRSA-positive

Xeonatal

Table

128

T. Mitsuda

et al.

neonates (0.9%) were positive for both MSSA and MRSA. The birth weight, duration of gestation and number of days in hospital of the 45 neonates who carried MRSA were: 3288.6 f450.8 g, 279.5 rf: 7.8 days and 6.9+ 1.6 days; and of the 367 neonates who were S. aUreU$ negative (controls) these values were 3 114.7 f 404.5 g, 277.7 + 9.0 days and 6.5 _+1.3 days, respectively (mean *SD). The prevalence of MRSA carriers decreased with time (Table I). Of the MRSA-positive neonates tested, 14.3% were still carriers at 12 months whereas 1.1% of the S. aureus-negative neonates had become MRSA positive. Thus at 12 months the prevalence of MRSA carriage was significantly higher in the MRSA-positive neonates vs. the neonates who were negative for S. aureus (P=O.O34). Isolation of MRSA f Yom the environment and health care providers MRSA was frequently isolated from the hospital environment between September 1991 and January 1992, but was not detected after February 1992. Serial examinations identified seven of 53 health care providers as carriers of MRSA. Only two were persistent MRSA carriers; five were transiently positive for MRSA. Epidemiological analysis of MRSA infection by conventional methods and PFGE At least three outbreaks of MRSA infection occurred in the hospital nursery between August 1991 and August 1992. To identify the origin of the strain of S. aweus obtained from each case, those from the 4.5 neonates were analysed by PFGE (Figure 1). Chromosomal DNA of each clone was digested by SmaI. DNA fragments were electrophoresed and the differences were evaluated according to the pattern of fragments. The genomic DNA fragmentation patterns were classified into three types: strains nos. l-19, and 21 as type I; strains nos. 24-26, 29, 30 and 33-37 as type II; strains 41-45 as type III. These findings supported the occurrence of three outbreaks of MRSA infection in the nursery. Results of the environmental study using PFGE are shown in Figure 2. Type II was commonly isolated from the floors of the ward and the nursery (strains nos. 2, 3, 8 and 11). These strains were also isolated from two nurses (N33) and (N39) (Figure 3). We identified transient MRSA carriers of epidemic types I or III, but no persistent carriers. The drainage area of the nursery bathtub (strain no. 1 in Figure 1) was contaminated with a type I MRSA strain. The epidemiological findings were supported by those from the coagulase-typing, enterotoxin-typing, and the test of TSST-1 production (Table II). Clinical j&dings in MRSA-positive infants In the 45 neonates who carried MRSA, skin pustules were found in the neck, axilla and inguinal region of eight patients, four patients had

Epidemiological

analysis

of MRSA

infection

in the nursery

129

600-

50-

(kb)

Figure 2. Pulsed-field gel electrophoresis uuwus (RIRSA) strains isolated from the top of the figure. Numbers l-11 represent Lambda (1) at the left top represents the concatemers).

analysis of 11 methicillin-resistant Staph~~lococcz~s environment. Date of isolation is indicated at the the MRSA strains isolated from the environment. molecular weight markers (lambda ladder DNA

130

600-

50-

Figure 3. Pulsed-field gel electrophoresis analysis of 10 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from health care providers. Date of isolation is indicated at the top of the figure. Each number represents the MRSA strains isolated from the individuals. Lambda (1) at the left top represents the molecular weight markers (lambda ladder DNA concatemers).

Epidemiological

analysis

of MRSA

infection

in the nursery

131

132

T. Mitsuda

et al.

conjunctivitis, one had an infected umbilical wound, and one had balanoposthitis. The pustules healed spontaneously without antibiotic therapy; the conjunctivitis was treated with ofloxacin eye drops, and the balanoposthitis with gentamicin ointment. The one-year follow-up of 18 of the 45 MRSA-positive infants showed that none of them had developed systemic infections. Discussion Conventional epidemiological methods useful for classifying S. UUY~US include coagulase-typing, enterotoxin-typing, phage-typing and proteasetyping employed in combination. Recent developments in molecular biology have also provided useful tools for evaluating epidemics of infectious disease. The combination of conventional and newer molecular biological techniques would be informative.12-‘6 Although advanced techniques are available, they may not be valid for epidemiological analysis if used alone. For example, poor plasmid stability (plasmid deletion) presents a problem when analysing plasmid profiles; 5% of S. LZUY~USlack plasmids.r7 The polymerase chain reaction (PCR) based on the fragmentation pattern of the coagulase gene and arbitrary PCR appear promising.1s~‘9 While PCR techniques are useful for rapid diagnosis, they are less precise than PFGE for typing strains of S. aureus, as the latter can distinguish some 31-37 strains.12,2*22 We evaluated three outbreaks of MRSA infection in the nursery during a 13 -month study. Despite efforts to disinfect the environment and educate health care providers, it was difficult to eliminate MRSA from the infants. While transient MRSA carriers disappeared from the health care providers, the resident MRSA carriers persisted. The practice of earlier rooming-in may encourage the growth of the normal resident flora of the skin and nasal mucosa of the neonates. It was impossible, to distinguish infection transmitted from the mother to the infant vs. nosocomial infection by clinical examination in the absence of epidemiological studies.“’ Our results suggest that the neonates had been contaminated with MRSA in the hospital environment. Our follow-up study showed that the incidence of the carriage decreased with age. Nevertheless, the incidence of MRSA carriage at one year was 14.3%, compared with 1 .l% in the control group. The weight of each infant enrolled in the study exceeded 1500 g, and all were delivered after 32 weeks of gestation. We did not encounter the lowbirth weight or premature infants whose immune defences are weak and may be particularly susceptible to infection by MRSA. No serious MRSA infections occurred in either infants or mothers during the three epidemics. There are several possible explanations for this. Firstly, the maternity hospital provides for normal delivery. It does not provide a neonatal intensive care facility. Secondly, antibiotic usage is restricted by the infection control committee in order to encourage the appropriate use of antibiotics.

Epidemiological

analysis

of MRSA

infection

in the nursery

133

Thirdly, most of the mothers and their infants are discharged within S-10 days of delivery, helping to reduce the incidence of exposure to MRSA during long-term admission. Twelve month follow-up showed a higher prevalence of MRSA carriers among the infants who had been MRSA positive in the neonatal period than among the control group. Thus, efforts must be taken to reduce the number of neonates who carry MRSA. The PFGE and conventional analyses indicated that the major route of MRSA infection in the nursery could be related to: (i) the health care providers who carried MRSA; and (ii) the routine bathing of the infants, in that the drainage area of the bathtubs was contaminated with MRSA. Infants are routinely bathed daily and the bathtubs are disinfected with 80% ethanol after every bath. Thus, the most likely source of the MRSA epidemics in this hospital was the health care providers. While we identified two MRSA resident carriers, their strains were not always those found in the epidemics. This indicates that any staff member could become a transient carrier of MRSA with a potential for starting an epidemic of MRSA. Education of health care providers and the maintenance of proper infection control procedures remain the most effective ways of preventing epidemics of MRSA. We thank Drs Yoshio Sumiyoshi, Toshiasa Suzuki, Hiroyuki Shigeta and Susumu Kimura, Department of Obstetrics and Gynecology, Yokohama City University School of Medicine for their assistance. This investigation was supported by a grant from Yokohama City for local specialized studies.

References 1. Dancer SJ, Noble WC. Nasal, axillary, and perineal carriage of Stuphylococcw UUY~US among u-omen: identification of strains producing epidermolytic toxin. J Cl& Pathol 1991; 44: 681-684. 2. Maple PA, Hamilton R!IJ, Brumfitt W. World-wide antibiotic resistance in methicillinresistant Staphylococctls ~UYCUS. Lancet 1989; I: 537-540. Staphylococcus UZLY~Z~S prevalence (editorial; 3. Boyce Jnl. Patterns of methicillin-resistant comment). Inject Control Hasp Epidemiol 1991; 12: 79-82. 4. Nozue J. Drug resistance of Staphylococcus uureus in the field of Obstetrics and Gynecology. Acta Ohst GynaecJpn 1989; 41: 449-453. 5. Tosaka WI, Yamane S, Okabe H. Isolation and antimicrobial susceptibilitv of methicillin resistant Staphylococcus uwew (l&IRSA) in Kumamoto LTniversitv Hospital. Jpn J Clip Med 1992; 50: 975-980. AH. E pi ‘d emit methicillin-gentamicin-resistant 6. Reboli AC, John JJ, Levkoff StaphylococczLs auzeus in a neonatal intensive care unit. Am J Dis Child 1989; 143: 3-l-39. 7. Webster J, Faoagali JL. Endemic methicillin-resistant Staphylococcus aUYeUs in a special care baby unit: a 2 year review. J Puediatr Child Health 1990; 26: 160-163. 8. nloore EP, Williams En’. A maternity hospital outbreak of methicillin-resistant Stuphylococczcs auseus. J Hosp Inject 1991; 19: S-16. 9. Carantonis LM, Spink MS. A selective salt egg agar medium for pathogenic Staphylococci. J Path Ract 1963; 86: 217-220. 10. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically-third edition; approved standard. *\JCCLS Doclrment M7-A3 1993.

134

T. Mitsuda

et al.

11. Smith CA, Cantor CR. Purification, specific fragmentation, and separation of large DNA molecules. Methods Enzvmol 1987: 155: 449-467. S, Ohta S, Shimokata K, Kato’N, Takeuchi J. Genomic DNA fingerprinting 12. Ichiyama by pulsed-field gel electrophoresis as an epidemiological marker for study of nosocomial infections caused by methicillin-resistant Staphylococcus aureus. J Clin Microbial 1991; 29: 2690-2695. K, Lenz W, Kayser FH, Shalit I, Krasemann C. Use of a ribosomal RNA 13. Hadorn gene probe for the epidemiological study of methicillin and ciprofloxacin resistant

Staphylococcus aureus. Eur 3’ Clin Microbial

Infect Dis 1990; 9: 649-653.

enzyme fragmentation 14. Coia JE, Noor HI, Platt DJ. Plasmid profiles and restriction patterns of plasmids of methicillin-sensitive and methicillin-resistant isolates of Staphylococcus aureus from hospital and the community. JMed Microbial 1988; 27: 271-276. BE, Ask E, Jenkins A, Hofstad T. DNA fingerprinting of isolates 15. Tveten Y, Kristiansen of Stanhylococcus aureus from newborns and their contacts. 7 Clin Microbial 1991; 29:

iioo-ii05

A, Bax R, Peerbooms P, Goessens WHF, van Leeuwen N, Quint WG. 16. Belkum Comparison of phage typing and DNA fingerprinting by polymerase chain reaction for discrimination of methicillin-resistant Stanhvlococcus aureus strains. ic Clin Microbial _ 1993; 31: 798-803. AJ, Roy I, Harding GP, Couperus JJ. Diversity and stability of restriction 17. Zuccarelli enzyme profiles of plasmid DNA from methicillin-resistant Staphylococcus aureus. J

Clin Microbial

1990; 28: 97-102.

typing of Staphylococcus aureus 18. Goh S-H, Byrne SK, Zhang JL, Chow AW. Molecular on the basis of coagulase gene polymorphisms. J Clin Microbial 1992; 30: 1642-I 645. P, Bourneix C, Prevost G, Andremont A. Random amplified polymorphic 19. Saulnier DNA assay is less discriminant than pulsed-field gel electrophoresis for typing strains of methicillin-resistant Staphylococcus aureus. J Clin Microbial 1993; 31: 982-985. B, Dahlet M, Bientz M, Mantz JM, Piemont Y. Pulsed-field 20. Prevost G, Pottecher gel electrophoresis as a new epidemiological tool for monitoring methicillin-resistant Staphylococcus aureus in an intensive care unit. J Hasp Infect 1991; 17: 255-269. by pulsed-field gel electrophoresis 21. Prevost G, Jaulhac B, Piemont Y. DNA fingerprinting is more effective than ribotyping in distinguishing among methicillin-resistant Staphylococcus aureus isolates. J Clin Microbial 1992; 30: 967-973. typing and 22. Struelens MJ, Deplano A, Godard C, Maes N, Serruys E. Epidemiological delineation of genetic relatedness of methicillin-resistant Staphylococcus aureus by macrorestriction analysis of genomic DNA by using pulsed-field gel electrophoresis. J

Chin Microbial

1992; 30: 2599-2605.