Accepted Manuscript Persistence of nasal colonization with human pathogenic bacteria and associated antimicrobial resistance in the German general population Robin Köck, Patrick Werner, Alexander W. Friedrich, Christian Fegeler, Karsten Becker, Prof. Dr. PII:
S2052-2975(15)00098-0
DOI:
10.1016/j.nmni.2015.11.004
Reference:
NMNI 97
To appear in:
New Microbes and New Infections
Received Date: 17 August 2015 Revised Date:
3 November 2015
Accepted Date: 4 November 2015
Please cite this article as: Köck R, Werner P, Friedrich AW, Fegeler C, Becker K, Prevalence of Multiresistant Microorganisms (PMM) Study Group, Persistence of nasal colonization with human pathogenic bacteria and associated antimicrobial resistance in the German general population, New Microbes and New Infections (2015), doi: 10.1016/j.nmni.2015.11.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Persistence of nasal colonization with human pathogenic bacteria and associated antimicrobial
2
resistance in the German general population
3 Robin Köck1, Patrick Werner2, Alexander W. Friedrich3, Christian Fegeler2, Karsten Becker1* and
5
Prevalence of Multiresistant Microorganisms (PMM) Study Group4
RI PT
4
6
1 University Hospital Münster, Institute of Medical Microbiology, 48149 Münster, Germany
8
2 University Heilbronn, GECKO Institute of Medicine, Informatics and Economy, 74081 Heilbronn,
9
Germany,
SC
7
3 University Hospital Groningen, Department for Medical Microbiology and Infection Control, 9700
11
Groningen, The Netherlands,
12
4 Prevalence of Multiresistant Microorganisms (PMM) Study Group: Olga Bindewald, Thanh T. Bui,
13
Claudia Eckhoff, Roland Epping, Lisa Kähmann, Monique Meurer, Jan Steger and Laura von
14
Auenmüller, Institute of Medical Microbiology, 48149 Münster, Germany
15
*Corresponding author: Domagkstr. 10; 48149 Münster, Germany, tel. +49 251 +83 55375; fax +49
16
251 83 52768, mail: [email protected]
19 20
TE D
EP
18
Running title: Bacterial colonization of the nares
AC C
17
M AN U
10
ACCEPTED MANUSCRIPT Abstract:
22
Background: The nares represent an important bacterial reservoir for endogenous infections. This
23
study aims to assess the prevalence of nasal colonization by different important pathogens, the
24
associated antimicrobial susceptibility and risk factors.
25
Methods: We performed a prospective cohort study among 1,878 non-hospitalized volunteers
26
recruited from the general population in Germany. Participants provided nasal swabs at three time-
27
points (each separated by 4-6 months). Staphylococcus aureus, Enterobacteriaceae and important
28
non-fermenters were cultured and subjected to susceptibility testing. Factors potentially influencing
29
bacterial colonization patterns were assessed.
30
Results: The overall prevalence of S. aureus, Enterobacteriaceae and non-fermenters was 41.0%,
31
33.4% and 3.7%, respectively. Thirteen participants (0.7%) were colonized with methicillin-resistant
32
S. aureus (MRSA). Enterobacteriaceae were mostly (>99%) susceptible against ciprofloxacin and
33
carbapenems (100%). Extended-Spectrum β-lactamase (ESBL) producing isolates were not detected
34
among K. oxytoca, K. pneumoniae and E. coli. Several lifestyle- and health-related factors (e.g.
35
household-size, travel, livestock-density of the residential area or occupational livestock contact,
36
atopic dermatitis, antidepressant or anti-infective drugs) were associated with colonization by
37
different microorganisms.
38
Conclusions: This study demonstrated unexpectedly high nasal colonization rates with
39
Enterobacteriaceae in the German general population, but rates of antibiotic resistance were low.
40
MRSA carriage was rare, but highly associated with occupational livestock contact.
42 43
SC
M AN U
TE D
EP
AC C
41
RI PT
21
Key words: nose; MRSA; ESBL; Germany; risk; livestock; pigs; urban; community
ACCEPTED MANUSCRIPT Introduction
45
Microorganisms colonizing the human body are involved in important immunological processes. They
46
prevent establishment of potentially harmful pathogens and assist in improving the immune system
47
[1]. On the other hand, the microbiota may promote the development of allergic diseases and is a
48
major reservoir for endogenous infections. For bacteria in the nasal habitat, the latter has mainly
49
been demonstrated for the role of nasal Staphylococcus aureus carriage in the development of
50
nosocomial infections such as bacteraemia, sternal or orthopaedic infections [2-5].
51
Recently, non-cultural methods have greatly increased knowledge on the communities of
52
microorganisms colonizing different body sites [6]. Hence, analyses of 16S rRNA sequences in
53
samples from the nares of healthy individuals demonstrated that >80% of rRNA sequences detected,
54
belonged to either Actinobacteria (e.g. cornyebacteria) or Firmicutes (e.g. staphylococci) [6]. In
55
addition, other bacterial taxa including Proteobacteria (e.g. Enterobacteriaceae or Pseudomonales),
56
Bacteriodetes or Fusobacteria were also found [6-8]. Interestingly, various studies indicated that the
57
composition of the microbiota of different human habitats is influenced by i.) several demographic
58
factors such as gender, age and ethnicity, but also ii.) by health-related factors like hospitalization,
59
intake of antibiotics, anti-pneumococcal vaccination or presence of viral infections and iii.) by
60
lifestyle-associated factors like frequency of hand washing or smoking [9-13].
61
Recognizing the value of culture-independent methods, these techniques have two major limitations.
62
First, they cannot assess bacterial antibiotic susceptibilities and second, the overload of information
63
on predominating phyla tends to dissolve information on the presence of important human
64
pathogens due to the small abundance of these species. However, it is important to overcome these
65
limitations as recent studies have indicated changes in the occurrence of antimicrobial resistant
66
pathogens in the healthy general population. Exemplarily, this was demonstrated for extended-
67
spectrum β–lactamase (ESBL) or carbapenemase producing Enterobacteriaceae and methicillin-
68
resistant S. aureus (MRSA), which were increasingly found as colonizers of the nares and the
69
intestinal tract in association with travel activities, contact with or ingestion of contaminated food
70
items or contact with livestock husbandries [14-18]. In addition, the emergence of antimicrobial
AC C
EP
TE D
M AN U
SC
RI PT
44
ACCEPTED MANUSCRIPT resistant Gram-negative bacteria among humans has mainly been documented by studies assessing
72
these pathogens in stool samples or rectal swabs. Data on their occurrence and persistence in the
73
nares, which might be important for the probability of transmission to the environment and to other
74
persons, are lacking.
75
Therefore, the objective of this prospective cohort study was to assess the occurrence of nasal
76
colonization of important facultative pathogenic bacteria including S. aureus, Enterobacteriaceae and
77
non-fermentative bacteria in samples from a large number of non-hospitalized volunteers. In
78
addition, information on associated antimicrobial resistance and lifestyle conditions was obtained
79
and correlated with the nasal bacterial colonization pattern.
M AN U
80
SC
RI PT
71
Methods
82
Non-hospitalized adult participants were recruited i.) in ambulatory departments of public health
83
offices (offering e.g. travel vaccination or health checks) or were employees of these offices, ii.) in
84
dentist practices, iii.) in a family physician’s practice and iv.) among students at colleges or
85
universities. At the beginning of the study, participants were informed about the study’s objectives
86
and provided written informed consent. Ethical approval was obtained by the ethical commission of
87
the Westphalian Wilhelms-University Münster (2006-268-f-S).
88
Samples were taken at three different time-points (June-August 2011, January-March 2012, August-
89
October 2012), each separated by at least four months. At every time-point, participants were asked
90
to provide a nasal swab and to answer a standardized questionnaire. Interviews and nasal swabs
91
were performed by trained medical or dental students. Answers of the participants were directly
92
assessed in a protected data-collection application portable on a USB flash drive.
93
From all participants pre-moistened nasal swabs (FLOQSwabs™, Copan, Murrieta, CA, USA) were
94
taken from the anterior nares. Swabs were immediately transferred to the microbiological
95
laboratory. After non-selective enrichment in Mueller-Hinton broth (24h, 36°C), swabs were streaked
96
onto Columbia blood agar and MacConkey agar as well as ESBL and S. aureus screening agars (all
97
Oxoid, Wesel, Germany) and incubated for 24 h at 36°C. All colonies suspicious for S. aureus,
AC C
EP
TE D
81
ACCEPTED MANUSCRIPT 98
Enterobacteriaceae or clinically important non-fermenters (i.e. Acinetobacter baumannii,
99
Pseudomonas sp., Achromobacter sp. and Stenotrophomonas sp.), respectively, were sub-cultured on Columbia blood agar.
101
For species identification, all isolates were tested using matrix-assisted laser desorption ionization
102
time-of-flight mass-spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was
103
performed using VITEK2 automated systems (and for some non-fermenter species using agar disc
104
diffusion) with clinical breakpoints and application of expert rules as recommended by the European
105
Committee on Antimicrobial Susceptibility Testing (EUCAST). All S. aureus isolates were analysed for
106
the presence of the nuc and mecA genes [19]. For S. aureus isolates that were cefoxitin-resistant,
107
mecC was tested in addition as previously described [20].
108
The questionnaire assessed the following data for each participant: year of birth, sex, ZIP code for
109
place of residence, country of birth, employment in healthcare institutions, employment with contact
110
to livestock animals, number of other persons living in the same household, living with companion
111
animals in the same household, being a smoker, having regular (i.e. at least weekly) contact to
112
medical personnel, having regular (i.e. at least weekly) contact to persons with employment in
113
livestock production, and travelling abroad (if yes, which countries; within the past 12 months). In
114
addition, participants were asked whether they suffered from diabetes mellitus, allergies, chronic
115
skin diseases (e.g. psoriasis, atopic dermatitis, acne), chronic diseases of the liver or the airways (e.g.
116
asthma, chronic obstructive pulmonary disease), chronic renal insufficiency, chronic inflammatory
117
diseases of the paranasal sinuses or the bowel, chronic osteomyelitis, solid tumors or haemato-
118
oncologic diseases. Participants provided information on whether they were immunocompromised
119
(if yes, why?), whether they had implanted devices (e.g. heart valves, prostheses). Moreover,
120
information on hospitalization (within the past 12 months) was obtained. Participants were asked
121
whether they took antibiotics or antimycotic agents (if yes, oral/intravenous or topical), oral
122
contraceptives or medication against clinical depression or neuroleptic agents. Data on density of
123
livestock and human population in different districts as well as geographical data were assessed from
124
the German Regional Database (https://www.regionalstatistik.de/; codes 116-33-4, 173-21-4, 449-
AC C
EP
TE D
M AN U
SC
RI PT
100
ACCEPTED MANUSCRIPT 01-4). Statistical analysis was performed using SPSS software (version 22). Chi-square or Fisher’s
126
exact tests were used to calculate differences between categorical variable; t-test was used for
127
parametric variables. P
S2052-2975(15)00098-0
DOI:
10.1016/j.nmni.2015.11.004
Reference:
NMNI 97
To appear in:
New Microbes and New Infections
Received Date: 17 August 2015 Revised Date:
3 November 2015
Accepted Date: 4 November 2015
Please cite this article as: Köck R, Werner P, Friedrich AW, Fegeler C, Becker K, Prevalence of Multiresistant Microorganisms (PMM) Study Group, Persistence of nasal colonization with human pathogenic bacteria and associated antimicrobial resistance in the German general population, New Microbes and New Infections (2015), doi: 10.1016/j.nmni.2015.11.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Persistence of nasal colonization with human pathogenic bacteria and associated antimicrobial
2
resistance in the German general population
3 Robin Köck1, Patrick Werner2, Alexander W. Friedrich3, Christian Fegeler2, Karsten Becker1* and
5
Prevalence of Multiresistant Microorganisms (PMM) Study Group4
RI PT
4
6
1 University Hospital Münster, Institute of Medical Microbiology, 48149 Münster, Germany
8
2 University Heilbronn, GECKO Institute of Medicine, Informatics and Economy, 74081 Heilbronn,
9
Germany,
SC
7
3 University Hospital Groningen, Department for Medical Microbiology and Infection Control, 9700
11
Groningen, The Netherlands,
12
4 Prevalence of Multiresistant Microorganisms (PMM) Study Group: Olga Bindewald, Thanh T. Bui,
13
Claudia Eckhoff, Roland Epping, Lisa Kähmann, Monique Meurer, Jan Steger and Laura von
14
Auenmüller, Institute of Medical Microbiology, 48149 Münster, Germany
15
*Corresponding author: Domagkstr. 10; 48149 Münster, Germany, tel. +49 251 +83 55375; fax +49
16
251 83 52768, mail: [email protected]
19 20
TE D
EP
18
Running title: Bacterial colonization of the nares
AC C
17
M AN U
10
ACCEPTED MANUSCRIPT Abstract:
22
Background: The nares represent an important bacterial reservoir for endogenous infections. This
23
study aims to assess the prevalence of nasal colonization by different important pathogens, the
24
associated antimicrobial susceptibility and risk factors.
25
Methods: We performed a prospective cohort study among 1,878 non-hospitalized volunteers
26
recruited from the general population in Germany. Participants provided nasal swabs at three time-
27
points (each separated by 4-6 months). Staphylococcus aureus, Enterobacteriaceae and important
28
non-fermenters were cultured and subjected to susceptibility testing. Factors potentially influencing
29
bacterial colonization patterns were assessed.
30
Results: The overall prevalence of S. aureus, Enterobacteriaceae and non-fermenters was 41.0%,
31
33.4% and 3.7%, respectively. Thirteen participants (0.7%) were colonized with methicillin-resistant
32
S. aureus (MRSA). Enterobacteriaceae were mostly (>99%) susceptible against ciprofloxacin and
33
carbapenems (100%). Extended-Spectrum β-lactamase (ESBL) producing isolates were not detected
34
among K. oxytoca, K. pneumoniae and E. coli. Several lifestyle- and health-related factors (e.g.
35
household-size, travel, livestock-density of the residential area or occupational livestock contact,
36
atopic dermatitis, antidepressant or anti-infective drugs) were associated with colonization by
37
different microorganisms.
38
Conclusions: This study demonstrated unexpectedly high nasal colonization rates with
39
Enterobacteriaceae in the German general population, but rates of antibiotic resistance were low.
40
MRSA carriage was rare, but highly associated with occupational livestock contact.
42 43
SC
M AN U
TE D
EP
AC C
41
RI PT
21
Key words: nose; MRSA; ESBL; Germany; risk; livestock; pigs; urban; community
ACCEPTED MANUSCRIPT Introduction
45
Microorganisms colonizing the human body are involved in important immunological processes. They
46
prevent establishment of potentially harmful pathogens and assist in improving the immune system
47
[1]. On the other hand, the microbiota may promote the development of allergic diseases and is a
48
major reservoir for endogenous infections. For bacteria in the nasal habitat, the latter has mainly
49
been demonstrated for the role of nasal Staphylococcus aureus carriage in the development of
50
nosocomial infections such as bacteraemia, sternal or orthopaedic infections [2-5].
51
Recently, non-cultural methods have greatly increased knowledge on the communities of
52
microorganisms colonizing different body sites [6]. Hence, analyses of 16S rRNA sequences in
53
samples from the nares of healthy individuals demonstrated that >80% of rRNA sequences detected,
54
belonged to either Actinobacteria (e.g. cornyebacteria) or Firmicutes (e.g. staphylococci) [6]. In
55
addition, other bacterial taxa including Proteobacteria (e.g. Enterobacteriaceae or Pseudomonales),
56
Bacteriodetes or Fusobacteria were also found [6-8]. Interestingly, various studies indicated that the
57
composition of the microbiota of different human habitats is influenced by i.) several demographic
58
factors such as gender, age and ethnicity, but also ii.) by health-related factors like hospitalization,
59
intake of antibiotics, anti-pneumococcal vaccination or presence of viral infections and iii.) by
60
lifestyle-associated factors like frequency of hand washing or smoking [9-13].
61
Recognizing the value of culture-independent methods, these techniques have two major limitations.
62
First, they cannot assess bacterial antibiotic susceptibilities and second, the overload of information
63
on predominating phyla tends to dissolve information on the presence of important human
64
pathogens due to the small abundance of these species. However, it is important to overcome these
65
limitations as recent studies have indicated changes in the occurrence of antimicrobial resistant
66
pathogens in the healthy general population. Exemplarily, this was demonstrated for extended-
67
spectrum β–lactamase (ESBL) or carbapenemase producing Enterobacteriaceae and methicillin-
68
resistant S. aureus (MRSA), which were increasingly found as colonizers of the nares and the
69
intestinal tract in association with travel activities, contact with or ingestion of contaminated food
70
items or contact with livestock husbandries [14-18]. In addition, the emergence of antimicrobial
AC C
EP
TE D
M AN U
SC
RI PT
44
ACCEPTED MANUSCRIPT resistant Gram-negative bacteria among humans has mainly been documented by studies assessing
72
these pathogens in stool samples or rectal swabs. Data on their occurrence and persistence in the
73
nares, which might be important for the probability of transmission to the environment and to other
74
persons, are lacking.
75
Therefore, the objective of this prospective cohort study was to assess the occurrence of nasal
76
colonization of important facultative pathogenic bacteria including S. aureus, Enterobacteriaceae and
77
non-fermentative bacteria in samples from a large number of non-hospitalized volunteers. In
78
addition, information on associated antimicrobial resistance and lifestyle conditions was obtained
79
and correlated with the nasal bacterial colonization pattern.
M AN U
80
SC
RI PT
71
Methods
82
Non-hospitalized adult participants were recruited i.) in ambulatory departments of public health
83
offices (offering e.g. travel vaccination or health checks) or were employees of these offices, ii.) in
84
dentist practices, iii.) in a family physician’s practice and iv.) among students at colleges or
85
universities. At the beginning of the study, participants were informed about the study’s objectives
86
and provided written informed consent. Ethical approval was obtained by the ethical commission of
87
the Westphalian Wilhelms-University Münster (2006-268-f-S).
88
Samples were taken at three different time-points (June-August 2011, January-March 2012, August-
89
October 2012), each separated by at least four months. At every time-point, participants were asked
90
to provide a nasal swab and to answer a standardized questionnaire. Interviews and nasal swabs
91
were performed by trained medical or dental students. Answers of the participants were directly
92
assessed in a protected data-collection application portable on a USB flash drive.
93
From all participants pre-moistened nasal swabs (FLOQSwabs™, Copan, Murrieta, CA, USA) were
94
taken from the anterior nares. Swabs were immediately transferred to the microbiological
95
laboratory. After non-selective enrichment in Mueller-Hinton broth (24h, 36°C), swabs were streaked
96
onto Columbia blood agar and MacConkey agar as well as ESBL and S. aureus screening agars (all
97
Oxoid, Wesel, Germany) and incubated for 24 h at 36°C. All colonies suspicious for S. aureus,
AC C
EP
TE D
81
ACCEPTED MANUSCRIPT 98
Enterobacteriaceae or clinically important non-fermenters (i.e. Acinetobacter baumannii,
99
Pseudomonas sp., Achromobacter sp. and Stenotrophomonas sp.), respectively, were sub-cultured on Columbia blood agar.
101
For species identification, all isolates were tested using matrix-assisted laser desorption ionization
102
time-of-flight mass-spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was
103
performed using VITEK2 automated systems (and for some non-fermenter species using agar disc
104
diffusion) with clinical breakpoints and application of expert rules as recommended by the European
105
Committee on Antimicrobial Susceptibility Testing (EUCAST). All S. aureus isolates were analysed for
106
the presence of the nuc and mecA genes [19]. For S. aureus isolates that were cefoxitin-resistant,
107
mecC was tested in addition as previously described [20].
108
The questionnaire assessed the following data for each participant: year of birth, sex, ZIP code for
109
place of residence, country of birth, employment in healthcare institutions, employment with contact
110
to livestock animals, number of other persons living in the same household, living with companion
111
animals in the same household, being a smoker, having regular (i.e. at least weekly) contact to
112
medical personnel, having regular (i.e. at least weekly) contact to persons with employment in
113
livestock production, and travelling abroad (if yes, which countries; within the past 12 months). In
114
addition, participants were asked whether they suffered from diabetes mellitus, allergies, chronic
115
skin diseases (e.g. psoriasis, atopic dermatitis, acne), chronic diseases of the liver or the airways (e.g.
116
asthma, chronic obstructive pulmonary disease), chronic renal insufficiency, chronic inflammatory
117
diseases of the paranasal sinuses or the bowel, chronic osteomyelitis, solid tumors or haemato-
118
oncologic diseases. Participants provided information on whether they were immunocompromised
119
(if yes, why?), whether they had implanted devices (e.g. heart valves, prostheses). Moreover,
120
information on hospitalization (within the past 12 months) was obtained. Participants were asked
121
whether they took antibiotics or antimycotic agents (if yes, oral/intravenous or topical), oral
122
contraceptives or medication against clinical depression or neuroleptic agents. Data on density of
123
livestock and human population in different districts as well as geographical data were assessed from
124
the German Regional Database (https://www.regionalstatistik.de/; codes 116-33-4, 173-21-4, 449-
AC C
EP
TE D
M AN U
SC
RI PT
100
ACCEPTED MANUSCRIPT 01-4). Statistical analysis was performed using SPSS software (version 22). Chi-square or Fisher’s
126
exact tests were used to calculate differences between categorical variable; t-test was used for
127
parametric variables. P
