JCM Accepted Manuscript Posted Online 27 May 2015 J. Clin. Microbiol. doi:10.1128/JCM.00470-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
JCM00470-15 Revision
1
Clinical and Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in a
2
Neonatal Intensive Care Unit in the Decade Following Implementation of an Active
3
Detection and Isolation Program
4 5 6
Melissa U. Nelson, MD;a* Matthew J. Bizzarro, MD;a Robert S. Baltimore, MD;b,c,d
7
Louise M. Dembry, MD;c,d,e, Patrick G. Gallagher, MDa,,f,g#
8 9
Division of Perinatal Medicine, Department of Pediatrics, Yale University School of
10
Medicine, New Haven, CT, USAa; Division of Infectious Disease, Department of
11
Pediatrics, Yale University School of Medicine, New Haven, CT, USAb; Department of
12
Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT,
13
USAc; Department of Hospital Epidemiology, Yale-New Haven Hospital, New Haven,
14
CT, USAd; Division of Infectious Disease, Department of Internal Medicine, Yale
15
University School of Medicine, New Haven, CT, USAe; Department of Pathology, Yale
16
University School of Medicine, New Haven, CT, USAf; Department of Genetics, Yale
17
University School of Medicine, New Haven, CT, USAg.
18 19
Running title: Clinical and Molecular Epidemiology of MRSA in a NICU
20 21
*Present affiliation: Melissa U. Nelson, Crouse Hospital and the Department of
22
Pediatrics at SUNY Upstate Medical University, Syracuse, NY, USA.
23
1
JCM00470-15 Revision
24
#Address correspondence to: Patrick G. Gallagher, Department of Pediatrics, Yale
25
University School of Medicine, 333 Cedar Street, P. O. Box 208064, New Haven, CT
26
06520-8064, USA. Tel: (203) 688-2896; Fax: (203) 785-6974; Email:
27
[email protected].
28 29
Presented in part: Pediatric Academic Societies Annual Meeting; Washington, D.C.;
30
May 2013.
31 32
2
JCM00470-15 Revision
33
ABSTRACT
34 35
Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent source of
36
infection in the neonatal intensive care unit (NICU), often associated with significant
37
morbidity. Active detection and isolation (ADI) programs aim to reduce transmission. We
38
describe a comprehensive analysis of the clinical and molecular epidemiology of MRSA
39
in an NICU between 2003-2013, in the decade following implementation of a MRSA ADI
40
program. Molecular analyses included strain typing by pulsed-field gel electrophoresis,
41
mec and accessory gene regulator group genotyping by multiplex PCR, and
42
identification of toxin and potential virulence factor genes via PCR-based assays. Of
43
8,387 neonates, 115 had MRSA colonization and/or infection (1.4%). The MRSA
44
colonization rate declined significantly during the study period from 2.2 to 0.5/1000
45
patient days (linear time, p=0.0003; quadratic time, p=0.006). There were nineteen
46
cases of MRSA infection (16.5%). Few epidemiologic or clinical differences were
47
identified between MRSA-colonized vs. MRSA-infected infants. Thirty-one different
48
strains of MRSA were identified with a shift from hospital-associated to combined
49
hospital- and community-associated strains over time. Panton-Valentine leukocidin-
50
positive USA300 strains caused five of the last eleven infections. SCCmec types II and
51
IVa and agr groups 1 and 2 were most predominant. One isolate possessed the gene
52
for toxic shock syndrome toxin; none had genes for exfoliative toxin A or B. These
53
results highlight recent trends in MRSA colonization and infection and the
54
corresponding changes in molecular epidemiology. Continued vigilance for this invasive
3
JCM00470-15 Revision
55
pathogen remains critical, and specific attention to the unique host, the neonate, and
56
the distinct environment, the NICU, is imperative.
4
JCM00470-15 Revision
57
Methicillin-resistant Staphylococcus aureus (MRSA) is a common etiology of life-
58
threatening, healthcare-associated infection in neonatal intensive care units (NICU)(1,
59
2). The National Nosocomial Infections Surveillance System observed a > 300%
60
increase in the incidence of late-onset MRSA infections in NICUs, from 0.7 to 3.1
61
infections/10,000 patient days between 1995 and 2004(3). Unique host and
62
environmental factors, including immature immune systems, high frequency of contact
63
with healthcare providers,(4) exposure to numerous invasive procedures, NICU over
64
crowding and understaffing,(5, 6) and prolonged hospitalization,(7) make NICU patients
65
at especially high risk of becoming colonized and infected with MRSA. Since
66
colonization with MRSA is a risk factor for development of MRSA infection, prevention
67
of MRSA transmission within the NICU is critical(7, 8). Many NICUs have implemented
68
active detection and isolation (ADI) programs, which involve surveillance to rapidly
69
identify affected patients, followed by cohorting and isolation with standard contact
70
precautions, in attempts to prevent MRSA transmission and reduce infection rates.
71
Several large NICUs have published reports regarding the clinical epidemiology
72
of neonatal MRSA following implementation of surveillance, transmission prevention,
73
and/or decolonization strategies(7, 9-12). Some also incorporated molecular analyses in
74
their studies, including antibiotic susceptibility testing,(9, 10) strain typing,(9, 11, 12)
75
genotyping,(9, 12), and/or Panton-Valentine leukocidin gene detection(9, 12). No
76
studies have comprehensively examined all of those molecular features of MRSA and
77
additionally assessed the presence or absence of genes encoding proteins for a variety
78
of staphylococcal toxins and potential virulence factors over an extensive time period
79
following implementation of an ADI program in a NICU.
5
JCM00470-15 Revision
80
In response to the appearance of MRSA in the NICU at Yale-New Haven
81
Children’s Hospital (YNHCH) in July 2002 and a cluster of MRSA infections between
82
November 2002 and February 2003 (Summary in Supplemental Data), a MRSA ADI
83
program with universal surveillance screening and isolation of MRSA-positive patients
84
was implemented. The objective of this study was to comprehensively analyze the
85
clinical and molecular epidemiology of MRSA colonization and infection during the ten
86
years following ADI program implementation.
87 88
6
JCM00470-15 Revision
89
MATERIALS AND METHODS
90 91
Study Design and Setting
92
This retrospective cohort study and laboratory evaluation took place in the
93
YNHCH NICU in New Haven, CT USA from March 1, 2003 to February 28, 2013 (Figure
94
1). The NICU is a 54-bed, level IV quaternary care referral center for neonates with
95
complex medical and surgical needs.
96
MRSA ADI Program
97
In March 2003, a MRSA ADI program was initiated in the NICU at YNHCH. Staff
98
education and training regarding active surveillance and infection control measures
99
occurred at time of program implementation. Surveillance nares cultures were obtained
100
at admission and weekly for every admitted patient. Patients with MRSA colonization or
101
infection were isolated and cohorted under standard contact precautions. No
102
decolonization strategies were employed.
103
Case Identification and Data Collection
104
A search of the Yale-New Haven Hospital (YNHH) microbiology database
105
identified every case of MRSA colonization and/or infection that occurred within the
106
NICU during the study period. Retrospective reviews of medical records were
107
conducted.
108
Definitions
109
MRSA colonization was defined as a positive MRSA surveillance nares culture.
110
MRSA infection was defined as a positive culture from blood, urine, CSF, wound,
111
tracheal aspirate, or other bodily fluid culture in the setting of clinical signs of infection
7
JCM00470-15 Revision
112
and subsequent treatment with appropriate antimicrobial therapy. A patient with multiple
113
MRSA-positive cultures was counted only once in the analyses. An outbreak was
114
defined as > 6 MRSA infections within a 12 month period. Prolonged rupture of
115
membranes,(13) chorioamnionitis,(13) bronchopulmonary dysplasia (BPD),(14)
116
necrotizing enterocolitis (NEC),(15) and late-onset sepsis were defined as
117
described(16).
118
MRSA Identification and Confirmation
119
Initially, MRSA surveillance screening specimens were cultured on Columbia 5%
120
sheep blood agar (Remel, Lenexa, KS USA) and colonies were identified as S. aureus
121
with the Staphaurex® (Remel, Lenexa, KS USA) rapid latex agglutination test.
122
Methicillin sensitivity versus methicillin resistance was initially determined by disk
123
diffusion with a 30-microgram cefoxitin (FOX) disk. Later, MRSA surveillance screening
124
was determined by SpectraTM MRSA® agar (Remel, Lenexa, KS USA), which is a
125
selective and differential chromogenic medium for MRSA detection. Multiplex PCR
126
confirmed presence of the mecA gene as described(17, 18).
127
Antibiotic Susceptibility Testing
128
Antibiotic susceptibility testing was performed by the disk diffusion method(19)
129
and later by an automated instrument system (VITEK® 2 System, bioMerieux, Durham,
130
NC USA)(20). Throughout the study period, there was variation in which antibiotics
131
were utilized for antibiotic susceptibility testing in the YNHH microbiology laboratory, so
132
strains were not uniformly subjected to testing with the same panel of antibiotics.
133
Strain Typing
8
JCM00470-15 Revision
134
Strain typing was performed by pulsed-field gel electrophoresis (PFGE) of SmaI-
135
digested bacterial DNA as described(21). Genomic DNA was prepared via Proteinase K
136
digestion of bacteria using standard methodology. Genomic DNA was digested with
137
SmaI restriction enzyme (Boehringer Mannheim, Indianapolis, IN USA) and separated
138
by electrophoresis with either a CHEF-DR II or CHEF-DR III apparatus (Bio-Rad,
139
Hercules, CA USA). Parameters included: temperature, 15°C; run time, 20 hours; switch
140
time, 5-40 seconds; and voltage, 198 V (CHEF-DR II) or 6 V/cm (CHEF-DR III). PFGE
141
patterns were compared with those from all other MRSA isolates observed at YNHH as
142
well as those from positive control strains representing the major circulating MRSA
143
clonal types. PFGE patterns that were identical without any band differences were
144
considered the same strain. PFGE patterns with 2 to 3 band differences were
145
considered closely related and subclassified as “strain-CR.” PFGE patterns with 4 to 6
146
band differences were considered possibly related and subclassified as “strain-PR,” and
147
PFGE patterns with >7 band differences were considered different. Unique strains were
148
categorized based on their serial identification at YNHH (i.e. First strain categorized as
149
1, second as 2, etc.).
150
Genotyping
151
Genomic DNA extraction and purification for use in PCR assays. MRSA isolates
152
were plated on BBLTM Columbia agar with 5% sheep blood (Beckton, Dickinson and
153
Company, Sparks, MD USA). Genomic DNA was extracted and purified from a single
154
colony grown from the isolate with MasterPureTM Gram Positive DNA Purification Kit
155
(epicentre, Madison, WI USA) according to the manufacturer’s instructions.
9
JCM00470-15 Revision
156
SCCmec typing. Multiplex PCR was utilized for SCCmec typing with primers
157
designed for SCCmec types and subtypes (Type I, II, III, IVa, IVb, IVc, IVd, V) and the
158
mecA gene, as described (Table 1)(18). PCR reaction mix included 2 μl of template
159
DNA in a 50-μl final reaction volume containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4),
160
2.5 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate, 2.0 units of Taq DNA
161
polymerase, and primer mix. The respective concentrations of primers were: 0.048 μM
162
of Type I primers, 0.032 μM of Type II primers, 0.04 μM of Type III primers, 0.104 μM of
163
Type IVa primers, 0.092 μM of Type IVb primers, 0.078 μM of Type IVc primers, 0.28
164
μM of Type IVd primers, 0.06 μM of Type V primers, and 0.046 μM of mecA primers.
165
Thermocycling conditions were 94°C for 5 min, followed by 10 cycles of 94°C for 45 s,
166
65°C for 45 s, and 72°C for 90 s, followed by 25 cycles of 94°C for 45 s, 55°C for 45 s,
167
and 72°C for 90 s, followed by 72°C for 10 minutes. The following S. aureus isolates
168
were provided by the NARSA program for use as control strains (Table 2): NRS 108
169
(SCCmec type I), NRS 382 (SCCmec type II), NRS 65 (SCCmec type III), NRS 384
170
(SCCmec type IVa), NRS 484 (SCCmec type IVc), NRS 385 (SCCmec type IVd). The
171
PCR amplicons were electrophoresed in a 2% agarose gel containing GelGreenTM
172
nucleic acid gel stain (Biotium, Hayward, CA USA) and visualized under ultraviolet light
173
as distinct bands corresponding to the expected molecular sizes (Table 1).
174
agr group designation. Multiplex PCR with primers designed to detect the four
175
different accessory gene regulator (agr) groups of the agr operon was performed as
176
described (Table 1)(22). PCR reaction mix included 2 μl of template DNA in a 50-μl final
177
reaction volume containing 0.03 μM of each primer (Pan, agr1, agr2, agr3, agr4).
178
Thermocycling conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 30 s,
10
JCM00470-15 Revision
179
60°C for 90 s, and 72°C for 60 s, followed by 68°C for 10 minutes. The following S.
180
aureus isolates were provided by the NARSA program for use as control strains (Table
181
2): NRS 385 (agr 1), NRS 382 (agr 2), NRS 123 (agr 3), NRS 166 (agr 4). The PCR
182
amplicons were electrophoresed in a 1% agarose gel containing GelGreenTM nucleic
183
acid gel stain (Biotium, Hayward, CA USA) and visualized under ultraviolet light as
184
distinct bands corresponding to the expected molecular sizes (Table 1).
185
Assessment for PVL. Multiplex PCR was performed with primers designed to
186
detect the Staphylococcus genus-specific 16S rRNA gene (internal positive control),
187
lukS/F-PV genes that encode for Panton-Valentine leukocidin (PVL), and the mecA
188
gene that confers methicillin resistance, as described (Table 1)(17). PCR reaction mix
189
included 2 μl of template DNA in a 50-μl final reaction volume containing 0.07 μM of
190
16S rRNA primers, 0.08 μM of lukS/F-PV primers, and 0.24 μM of mecA primers.
191
Thermocycling conditions were set at 94°C for 10 min, followed by 10 cycles of 94°C for
192
45 s, 55°C for 45 s, and 72°C for 75 s, followed by 25 cycles of 94°C for 45 s, 50°C for
193
45 s, and 72°C for 75 s. The following S. aureus isolates were used as control strains
194
(Table 2): ATCC 29213 (PVL-negative MSSA), ATCC 25923 (PVL-positive MSSA), and
195
ATCC 33591 (PVL-negative MRSA). The PCR amplicons were electrophoresed in a 2%
196
agarose gel containing GelGreenTM nucleic acid gel stain (Biotium, Hayward, CA USA)
197
and visualized under ultraviolet light as distinct bands corresponding to the expected
198
molecular sizes (Table 1).
199
Assessment for ACME. Multiplex PCR with primers designed to detect the arcA
200
gene on the arginine catabolic mobile element (ACME), which is a genetic element
201
specific to USA300, was performed as described (Table 1)(23). PCR reaction mix
11
JCM00470-15 Revision
202
included 2 μl of template DNA in a 50-μl final reaction volume containing 0.3 μM of arcA
203
primers. Thermocycling conditions were set at 94°C for 4 min, followed by 10 cycles of
204
94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, followed by 25 cycles of 94°C for 30 s,
205
52°C for 30 s, and 72°C for 45 s. The following S. aureus isolate was provided by the
206
NARSA Program for use as a control strain (Table 2): NRS 384 (USA300). The PCR
207
amplicons were electrophoresed in a 2% agarose gel containing GelGreenTM nucleic
208
acid gel stain (Biotium, Hayward, CA USA) and visualized under ultraviolet light as a
209
distinct band corresponding to the expected molecular size of 513 bp (Table 1).
210
Assessment for TSST-1, ETA, and ETB. Multiplex PCR with primers designed to
211
detect the toxic shock syndrome toxin (TSST-1) and the exfoliative toxins, Exfoliative
212
toxin A (ETA) and Exfoliative toxin B (ETB), was performed as described (Table 1)(24).
213
PCR reaction mix included 2 μl of template DNA in a 50-μl final reaction volume
214
containing 500 mM KCl, 100 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.2 mM of each
215
deoxynucleoside triphosphate, 2.5 units of Taq DNA polymerase, and primer mix. The
216
respective concentrations of primers were: 50 pmol of eta primers, 20 pmol etb primers,
217
and 20 pmol of tst primers. The following S. aureus isolates were provided by the
218
NARSA Program for use as control strains (Table 2): NRS 383 (TSST-1-positive), NRS
219
167 (ETA-positive), NRS 165 (ETA-positive, ETB-positive). The PCR amplicons were
220
electrophoresed in a 2% agarose gel containing GelGreenTM nucleic acid gel stain
221
(Biotium, Hayward, CA USA) and visualized under ultraviolet light as distinct bands
222
corresponding to the expected molecular sizes (Table 1).
223 224
Assessment for LukE-LukD leukocidin. PCR with primers designed to detect the lukE-lukD gene that encodes for LukE-LukD leukocidin was performed (Table 1)(25).
12
JCM00470-15 Revision
225
PCR reaction mix included 2 μl of template DNA in a 50-μl final reaction volume
226
containing 0.2 μM of each lukE-lukD primer. Thermocycling conditions were set at 94°C
227
for 30 s, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s,
228
followed by 72°C for 60 s. The following S. aureus isolates were provided by the
229
NARSA Program for use as control strains (Table 2): NRS 164 (LukE-LukD-positive),
230
NRS 165 (LukE-LukD-positive), NRS 166 (LukE-LukD-positive), NRS 167 (LukE-LukD-
231
negative). The PCR amplicons were electrophoresed in a 2% agarose gel containing
232
GelGreenTM nucleic acid gel stain (Biotium, Hayward, CA USA) and visualized under
233
ultraviolet light as a distinct band corresponding to the expected molecular size of 269
234
bp (Table 1).
235
Assessment for beta-hemolysin. PCR with primers designed to detect the hlb
236
gene that encodes for beta-hemolysin was performed (Table 1)(25). PCR reaction mix
237
included 2 μl of template DNA in a 50-μl final reaction volume containing 0.2 μM of each
238
lukE-lukD primer. Thermocycling conditions were set at 94°C for 30 s, followed by 45
239
cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 72°C for 60 s. The
240
PCR amplicons were electrophoresed in a 2% agarose gel containing GelGreenTM
241
nucleic acid gel stain (Biotium, Hayward, CA USA) and visualized under ultraviolet light
242
as a distinct band corresponding to the expected molecular size of 309 bp (Table 1).
243
Statistical Analysis
244
Univariate comparisons of continuous data were made utilizing the independent
245
samples student’s t-test. Dichotomous data were compared using Pearson’s Chi-square
246
or Fisher’s exact test for any cell containing
JCM00470-15 Revision
1
Clinical and Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in a
2
Neonatal Intensive Care Unit in the Decade Following Implementation of an Active
3
Detection and Isolation Program
4 5 6
Melissa U. Nelson, MD;a* Matthew J. Bizzarro, MD;a Robert S. Baltimore, MD;b,c,d
7
Louise M. Dembry, MD;c,d,e, Patrick G. Gallagher, MDa,,f,g#
8 9
Division of Perinatal Medicine, Department of Pediatrics, Yale University School of
10
Medicine, New Haven, CT, USAa; Division of Infectious Disease, Department of
11
Pediatrics, Yale University School of Medicine, New Haven, CT, USAb; Department of
12
Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT,
13
USAc; Department of Hospital Epidemiology, Yale-New Haven Hospital, New Haven,
14
CT, USAd; Division of Infectious Disease, Department of Internal Medicine, Yale
15
University School of Medicine, New Haven, CT, USAe; Department of Pathology, Yale
16
University School of Medicine, New Haven, CT, USAf; Department of Genetics, Yale
17
University School of Medicine, New Haven, CT, USAg.
18 19
Running title: Clinical and Molecular Epidemiology of MRSA in a NICU
20 21
*Present affiliation: Melissa U. Nelson, Crouse Hospital and the Department of
22
Pediatrics at SUNY Upstate Medical University, Syracuse, NY, USA.
23
1
JCM00470-15 Revision
24
#Address correspondence to: Patrick G. Gallagher, Department of Pediatrics, Yale
25
University School of Medicine, 333 Cedar Street, P. O. Box 208064, New Haven, CT
26
06520-8064, USA. Tel: (203) 688-2896; Fax: (203) 785-6974; Email:
27
[email protected].
28 29
Presented in part: Pediatric Academic Societies Annual Meeting; Washington, D.C.;
30
May 2013.
31 32
2
JCM00470-15 Revision
33
ABSTRACT
34 35
Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent source of
36
infection in the neonatal intensive care unit (NICU), often associated with significant
37
morbidity. Active detection and isolation (ADI) programs aim to reduce transmission. We
38
describe a comprehensive analysis of the clinical and molecular epidemiology of MRSA
39
in an NICU between 2003-2013, in the decade following implementation of a MRSA ADI
40
program. Molecular analyses included strain typing by pulsed-field gel electrophoresis,
41
mec and accessory gene regulator group genotyping by multiplex PCR, and
42
identification of toxin and potential virulence factor genes via PCR-based assays. Of
43
8,387 neonates, 115 had MRSA colonization and/or infection (1.4%). The MRSA
44
colonization rate declined significantly during the study period from 2.2 to 0.5/1000
45
patient days (linear time, p=0.0003; quadratic time, p=0.006). There were nineteen
46
cases of MRSA infection (16.5%). Few epidemiologic or clinical differences were
47
identified between MRSA-colonized vs. MRSA-infected infants. Thirty-one different
48
strains of MRSA were identified with a shift from hospital-associated to combined
49
hospital- and community-associated strains over time. Panton-Valentine leukocidin-
50
positive USA300 strains caused five of the last eleven infections. SCCmec types II and
51
IVa and agr groups 1 and 2 were most predominant. One isolate possessed the gene
52
for toxic shock syndrome toxin; none had genes for exfoliative toxin A or B. These
53
results highlight recent trends in MRSA colonization and infection and the
54
corresponding changes in molecular epidemiology. Continued vigilance for this invasive
3
JCM00470-15 Revision
55
pathogen remains critical, and specific attention to the unique host, the neonate, and
56
the distinct environment, the NICU, is imperative.
4
JCM00470-15 Revision
57
Methicillin-resistant Staphylococcus aureus (MRSA) is a common etiology of life-
58
threatening, healthcare-associated infection in neonatal intensive care units (NICU)(1,
59
2). The National Nosocomial Infections Surveillance System observed a > 300%
60
increase in the incidence of late-onset MRSA infections in NICUs, from 0.7 to 3.1
61
infections/10,000 patient days between 1995 and 2004(3). Unique host and
62
environmental factors, including immature immune systems, high frequency of contact
63
with healthcare providers,(4) exposure to numerous invasive procedures, NICU over
64
crowding and understaffing,(5, 6) and prolonged hospitalization,(7) make NICU patients
65
at especially high risk of becoming colonized and infected with MRSA. Since
66
colonization with MRSA is a risk factor for development of MRSA infection, prevention
67
of MRSA transmission within the NICU is critical(7, 8). Many NICUs have implemented
68
active detection and isolation (ADI) programs, which involve surveillance to rapidly
69
identify affected patients, followed by cohorting and isolation with standard contact
70
precautions, in attempts to prevent MRSA transmission and reduce infection rates.
71
Several large NICUs have published reports regarding the clinical epidemiology
72
of neonatal MRSA following implementation of surveillance, transmission prevention,
73
and/or decolonization strategies(7, 9-12). Some also incorporated molecular analyses in
74
their studies, including antibiotic susceptibility testing,(9, 10) strain typing,(9, 11, 12)
75
genotyping,(9, 12), and/or Panton-Valentine leukocidin gene detection(9, 12). No
76
studies have comprehensively examined all of those molecular features of MRSA and
77
additionally assessed the presence or absence of genes encoding proteins for a variety
78
of staphylococcal toxins and potential virulence factors over an extensive time period
79
following implementation of an ADI program in a NICU.
5
JCM00470-15 Revision
80
In response to the appearance of MRSA in the NICU at Yale-New Haven
81
Children’s Hospital (YNHCH) in July 2002 and a cluster of MRSA infections between
82
November 2002 and February 2003 (Summary in Supplemental Data), a MRSA ADI
83
program with universal surveillance screening and isolation of MRSA-positive patients
84
was implemented. The objective of this study was to comprehensively analyze the
85
clinical and molecular epidemiology of MRSA colonization and infection during the ten
86
years following ADI program implementation.
87 88
6
JCM00470-15 Revision
89
MATERIALS AND METHODS
90 91
Study Design and Setting
92
This retrospective cohort study and laboratory evaluation took place in the
93
YNHCH NICU in New Haven, CT USA from March 1, 2003 to February 28, 2013 (Figure
94
1). The NICU is a 54-bed, level IV quaternary care referral center for neonates with
95
complex medical and surgical needs.
96
MRSA ADI Program
97
In March 2003, a MRSA ADI program was initiated in the NICU at YNHCH. Staff
98
education and training regarding active surveillance and infection control measures
99
occurred at time of program implementation. Surveillance nares cultures were obtained
100
at admission and weekly for every admitted patient. Patients with MRSA colonization or
101
infection were isolated and cohorted under standard contact precautions. No
102
decolonization strategies were employed.
103
Case Identification and Data Collection
104
A search of the Yale-New Haven Hospital (YNHH) microbiology database
105
identified every case of MRSA colonization and/or infection that occurred within the
106
NICU during the study period. Retrospective reviews of medical records were
107
conducted.
108
Definitions
109
MRSA colonization was defined as a positive MRSA surveillance nares culture.
110
MRSA infection was defined as a positive culture from blood, urine, CSF, wound,
111
tracheal aspirate, or other bodily fluid culture in the setting of clinical signs of infection
7
JCM00470-15 Revision
112
and subsequent treatment with appropriate antimicrobial therapy. A patient with multiple
113
MRSA-positive cultures was counted only once in the analyses. An outbreak was
114
defined as > 6 MRSA infections within a 12 month period. Prolonged rupture of
115
membranes,(13) chorioamnionitis,(13) bronchopulmonary dysplasia (BPD),(14)
116
necrotizing enterocolitis (NEC),(15) and late-onset sepsis were defined as
117
described(16).
118
MRSA Identification and Confirmation
119
Initially, MRSA surveillance screening specimens were cultured on Columbia 5%
120
sheep blood agar (Remel, Lenexa, KS USA) and colonies were identified as S. aureus
121
with the Staphaurex® (Remel, Lenexa, KS USA) rapid latex agglutination test.
122
Methicillin sensitivity versus methicillin resistance was initially determined by disk
123
diffusion with a 30-microgram cefoxitin (FOX) disk. Later, MRSA surveillance screening
124
was determined by SpectraTM MRSA® agar (Remel, Lenexa, KS USA), which is a
125
selective and differential chromogenic medium for MRSA detection. Multiplex PCR
126
confirmed presence of the mecA gene as described(17, 18).
127
Antibiotic Susceptibility Testing
128
Antibiotic susceptibility testing was performed by the disk diffusion method(19)
129
and later by an automated instrument system (VITEK® 2 System, bioMerieux, Durham,
130
NC USA)(20). Throughout the study period, there was variation in which antibiotics
131
were utilized for antibiotic susceptibility testing in the YNHH microbiology laboratory, so
132
strains were not uniformly subjected to testing with the same panel of antibiotics.
133
Strain Typing
8
JCM00470-15 Revision
134
Strain typing was performed by pulsed-field gel electrophoresis (PFGE) of SmaI-
135
digested bacterial DNA as described(21). Genomic DNA was prepared via Proteinase K
136
digestion of bacteria using standard methodology. Genomic DNA was digested with
137
SmaI restriction enzyme (Boehringer Mannheim, Indianapolis, IN USA) and separated
138
by electrophoresis with either a CHEF-DR II or CHEF-DR III apparatus (Bio-Rad,
139
Hercules, CA USA). Parameters included: temperature, 15°C; run time, 20 hours; switch
140
time, 5-40 seconds; and voltage, 198 V (CHEF-DR II) or 6 V/cm (CHEF-DR III). PFGE
141
patterns were compared with those from all other MRSA isolates observed at YNHH as
142
well as those from positive control strains representing the major circulating MRSA
143
clonal types. PFGE patterns that were identical without any band differences were
144
considered the same strain. PFGE patterns with 2 to 3 band differences were
145
considered closely related and subclassified as “strain-CR.” PFGE patterns with 4 to 6
146
band differences were considered possibly related and subclassified as “strain-PR,” and
147
PFGE patterns with >7 band differences were considered different. Unique strains were
148
categorized based on their serial identification at YNHH (i.e. First strain categorized as
149
1, second as 2, etc.).
150
Genotyping
151
Genomic DNA extraction and purification for use in PCR assays. MRSA isolates
152
were plated on BBLTM Columbia agar with 5% sheep blood (Beckton, Dickinson and
153
Company, Sparks, MD USA). Genomic DNA was extracted and purified from a single
154
colony grown from the isolate with MasterPureTM Gram Positive DNA Purification Kit
155
(epicentre, Madison, WI USA) according to the manufacturer’s instructions.
9
JCM00470-15 Revision
156
SCCmec typing. Multiplex PCR was utilized for SCCmec typing with primers
157
designed for SCCmec types and subtypes (Type I, II, III, IVa, IVb, IVc, IVd, V) and the
158
mecA gene, as described (Table 1)(18). PCR reaction mix included 2 μl of template
159
DNA in a 50-μl final reaction volume containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4),
160
2.5 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate, 2.0 units of Taq DNA
161
polymerase, and primer mix. The respective concentrations of primers were: 0.048 μM
162
of Type I primers, 0.032 μM of Type II primers, 0.04 μM of Type III primers, 0.104 μM of
163
Type IVa primers, 0.092 μM of Type IVb primers, 0.078 μM of Type IVc primers, 0.28
164
μM of Type IVd primers, 0.06 μM of Type V primers, and 0.046 μM of mecA primers.
165
Thermocycling conditions were 94°C for 5 min, followed by 10 cycles of 94°C for 45 s,
166
65°C for 45 s, and 72°C for 90 s, followed by 25 cycles of 94°C for 45 s, 55°C for 45 s,
167
and 72°C for 90 s, followed by 72°C for 10 minutes. The following S. aureus isolates
168
were provided by the NARSA program for use as control strains (Table 2): NRS 108
169
(SCCmec type I), NRS 382 (SCCmec type II), NRS 65 (SCCmec type III), NRS 384
170
(SCCmec type IVa), NRS 484 (SCCmec type IVc), NRS 385 (SCCmec type IVd). The
171
PCR amplicons were electrophoresed in a 2% agarose gel containing GelGreenTM
172
nucleic acid gel stain (Biotium, Hayward, CA USA) and visualized under ultraviolet light
173
as distinct bands corresponding to the expected molecular sizes (Table 1).
174
agr group designation. Multiplex PCR with primers designed to detect the four
175
different accessory gene regulator (agr) groups of the agr operon was performed as
176
described (Table 1)(22). PCR reaction mix included 2 μl of template DNA in a 50-μl final
177
reaction volume containing 0.03 μM of each primer (Pan, agr1, agr2, agr3, agr4).
178
Thermocycling conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 30 s,
10
JCM00470-15 Revision
179
60°C for 90 s, and 72°C for 60 s, followed by 68°C for 10 minutes. The following S.
180
aureus isolates were provided by the NARSA program for use as control strains (Table
181
2): NRS 385 (agr 1), NRS 382 (agr 2), NRS 123 (agr 3), NRS 166 (agr 4). The PCR
182
amplicons were electrophoresed in a 1% agarose gel containing GelGreenTM nucleic
183
acid gel stain (Biotium, Hayward, CA USA) and visualized under ultraviolet light as
184
distinct bands corresponding to the expected molecular sizes (Table 1).
185
Assessment for PVL. Multiplex PCR was performed with primers designed to
186
detect the Staphylococcus genus-specific 16S rRNA gene (internal positive control),
187
lukS/F-PV genes that encode for Panton-Valentine leukocidin (PVL), and the mecA
188
gene that confers methicillin resistance, as described (Table 1)(17). PCR reaction mix
189
included 2 μl of template DNA in a 50-μl final reaction volume containing 0.07 μM of
190
16S rRNA primers, 0.08 μM of lukS/F-PV primers, and 0.24 μM of mecA primers.
191
Thermocycling conditions were set at 94°C for 10 min, followed by 10 cycles of 94°C for
192
45 s, 55°C for 45 s, and 72°C for 75 s, followed by 25 cycles of 94°C for 45 s, 50°C for
193
45 s, and 72°C for 75 s. The following S. aureus isolates were used as control strains
194
(Table 2): ATCC 29213 (PVL-negative MSSA), ATCC 25923 (PVL-positive MSSA), and
195
ATCC 33591 (PVL-negative MRSA). The PCR amplicons were electrophoresed in a 2%
196
agarose gel containing GelGreenTM nucleic acid gel stain (Biotium, Hayward, CA USA)
197
and visualized under ultraviolet light as distinct bands corresponding to the expected
198
molecular sizes (Table 1).
199
Assessment for ACME. Multiplex PCR with primers designed to detect the arcA
200
gene on the arginine catabolic mobile element (ACME), which is a genetic element
201
specific to USA300, was performed as described (Table 1)(23). PCR reaction mix
11
JCM00470-15 Revision
202
included 2 μl of template DNA in a 50-μl final reaction volume containing 0.3 μM of arcA
203
primers. Thermocycling conditions were set at 94°C for 4 min, followed by 10 cycles of
204
94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, followed by 25 cycles of 94°C for 30 s,
205
52°C for 30 s, and 72°C for 45 s. The following S. aureus isolate was provided by the
206
NARSA Program for use as a control strain (Table 2): NRS 384 (USA300). The PCR
207
amplicons were electrophoresed in a 2% agarose gel containing GelGreenTM nucleic
208
acid gel stain (Biotium, Hayward, CA USA) and visualized under ultraviolet light as a
209
distinct band corresponding to the expected molecular size of 513 bp (Table 1).
210
Assessment for TSST-1, ETA, and ETB. Multiplex PCR with primers designed to
211
detect the toxic shock syndrome toxin (TSST-1) and the exfoliative toxins, Exfoliative
212
toxin A (ETA) and Exfoliative toxin B (ETB), was performed as described (Table 1)(24).
213
PCR reaction mix included 2 μl of template DNA in a 50-μl final reaction volume
214
containing 500 mM KCl, 100 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.2 mM of each
215
deoxynucleoside triphosphate, 2.5 units of Taq DNA polymerase, and primer mix. The
216
respective concentrations of primers were: 50 pmol of eta primers, 20 pmol etb primers,
217
and 20 pmol of tst primers. The following S. aureus isolates were provided by the
218
NARSA Program for use as control strains (Table 2): NRS 383 (TSST-1-positive), NRS
219
167 (ETA-positive), NRS 165 (ETA-positive, ETB-positive). The PCR amplicons were
220
electrophoresed in a 2% agarose gel containing GelGreenTM nucleic acid gel stain
221
(Biotium, Hayward, CA USA) and visualized under ultraviolet light as distinct bands
222
corresponding to the expected molecular sizes (Table 1).
223 224
Assessment for LukE-LukD leukocidin. PCR with primers designed to detect the lukE-lukD gene that encodes for LukE-LukD leukocidin was performed (Table 1)(25).
12
JCM00470-15 Revision
225
PCR reaction mix included 2 μl of template DNA in a 50-μl final reaction volume
226
containing 0.2 μM of each lukE-lukD primer. Thermocycling conditions were set at 94°C
227
for 30 s, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s,
228
followed by 72°C for 60 s. The following S. aureus isolates were provided by the
229
NARSA Program for use as control strains (Table 2): NRS 164 (LukE-LukD-positive),
230
NRS 165 (LukE-LukD-positive), NRS 166 (LukE-LukD-positive), NRS 167 (LukE-LukD-
231
negative). The PCR amplicons were electrophoresed in a 2% agarose gel containing
232
GelGreenTM nucleic acid gel stain (Biotium, Hayward, CA USA) and visualized under
233
ultraviolet light as a distinct band corresponding to the expected molecular size of 269
234
bp (Table 1).
235
Assessment for beta-hemolysin. PCR with primers designed to detect the hlb
236
gene that encodes for beta-hemolysin was performed (Table 1)(25). PCR reaction mix
237
included 2 μl of template DNA in a 50-μl final reaction volume containing 0.2 μM of each
238
lukE-lukD primer. Thermocycling conditions were set at 94°C for 30 s, followed by 45
239
cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 72°C for 60 s. The
240
PCR amplicons were electrophoresed in a 2% agarose gel containing GelGreenTM
241
nucleic acid gel stain (Biotium, Hayward, CA USA) and visualized under ultraviolet light
242
as a distinct band corresponding to the expected molecular size of 309 bp (Table 1).
243
Statistical Analysis
244
Univariate comparisons of continuous data were made utilizing the independent
245
samples student’s t-test. Dichotomous data were compared using Pearson’s Chi-square
246
or Fisher’s exact test for any cell containing
