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Journal ListAccess Microbiolv.2(1); 2020PMC7525055

Logo of accessmicro
Access Microbiol. 2020; 2(1): acmi000077.
Published online 2019 Nov 5. doi: 10.1099/acmi.0.000077
PMCID: PMC7525055
PMID: 33062936
Prevalence and mechanisms of fluoroquinolone-resistant Escherichia coli among sheltered
companion animals
Kaoru Umeda, 1 ,* Atsushi Hase, 1 Akira Fukuda, 1 Masashi Matsuo, 2 Tomoaki Horimoto, 2 and
Jun Ogasawara 1
Author information Article notes Copyright and License information Disclaimer
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Abstract
To better understand the prevalence of fluoroquinolone-resistant Escherichia coli among
sheltered companion animals, we conducted a screening study of 38 dogs and 78 cats and
investigated the resistance mechanisms and characteristics of the isolates.
Fluoroquinolone-resistant E. coli was detected in 18 dogs (47.4 %) and 14 cats (17.9 %). The
isolates carried one to four mutations in the gyrA, parC and parE genes of the quinolone
resistance-determining region, and the number of mutations was proportional to the MIC for
ciprofloxacin. For plasmid-mediated quinolone resistance, aac-(6 ′)-Ib-cr was detected in nine
isolates, qnrS in five isolates and qnrB in one isolate. A relationship between the presence of
these genes and MIC for ciprofloxacin was not apparent. Statistical analysis indicated that
fluoroquinolone-resistant E. coli was widely distributed among sheltered companion animals
with various attributes. This may relate to the wide dissemination of fluoroquinolone resistance
among humans and other animals in Japan.

Keywords: fluoroquinolone-resistant Escherichia coli, companion animals, QRDR, PMQR

Go to:
Data Summary
This paper did not require other supporting external data.

Impact Statement
Fluoroquinolones are broad-spectrum antimicrobials that are useful for the treatment of
bacterial infections. However, fluoroquinolone resistance has increased in the clinical field. This
paper first clarified that fluoroquinolone-resistant Escherichia coli was widely distributed among
sheltered companion animals with a wide range of individual attributes. This finding is
important with regard to the circulation of fluoroquinolone resistance among humans, among
animals and in the environment. It may have considerable influence on the treatment of
infectious disease in both animals and humans given that 46.9 % of isolates were classified as
multidrug-resistant bacteria. In order to lower the overall level of fluoroquinolone resistance, it
will be necessary to reduce fluoroquinolone use and continue surveillance not only in the human
clinical field but also in the veterinary field.

Go to:
Introduction
Fluoroquinolones are broad-spectrum and effective bactericidal antimicrobials that have been
approved for use in the treatment of various bacterial infections. Recently, fluoroquinolone
resistance has increased in the clinical field nationwide [1]. The Japan Nosocomial Infections
Surveillance (JANIS) reported that 60.2 and 59.0 % of Escherichia coli isolates from inpatient
specimens obtained in 2015 and 2016, respectively, were resistant to levofloxacin [2]. The main
fluoroquinolone resistance mechanism is the chromosomal mutation of quinolone
resistance-determining region (QRDR) in DNA gyrase and topoisomerase IV. Another
mechanism, plasmid-mediated quinolone resistance (PMQR), was also found to contribute to
quinolone resistance [3–5].

We have previously reported the prevalence of cephalosporin-resistant Enterobacteriaceae

among sheltered companion animals; 14.6 % of dogs and 11.0 % of cats harboured
cephalosporin-resistant bacteria, and 33 % of these isolates were also resistant to
fluoroquinolone. Based on the distribution of several types of antimicrobial-resistant bacteria
among animals with a wide range of individual attributes, we concluded that companion animals
may play a bridging role in the circulation of antimicrobial-resistant bacteria from humans and
other origins [6]. Therefore, surveillance of sheltered companion animals may be useful in
determining the prevalence of antimicrobial-resistant bacteria.
Several studies have demonstrated the presence and characteristics of fluoroquinolone-resistant
E. coli among companion animals [7–11]. However, most of them included results from clinical
isolates obtained from animal hospitals and did not aim to demonstrate the prevalence of
fluoroquinolone-resistant bacteria among a wide range of companion animals.

Based on the above background, we first established a means to understand the prevalence of
fluoroquinolone-resistant E. coli among diverse dogs and cats, and then screened a number of
sheltered animals and investigated the resistance mechanisms and genetic characteristics of the
isolates.

Go to:
Methods
Faecal samples or rectal swabs were collected between April 2016 and January 2017 from 38 dogs
and 78 cats brought to Osaka Municipal Animal Care and Control Center. The sampling and
classifying of animals by individual attributes were done as in our previous report [6]. Samples
were inoculated on MacConkey agar (Nissui Pharmaceutical), supplemented with ciprofloxacin
at 4 mg l−1, and incubated at 37 °C for 20 h. Subculturing was performed on up to five E. coli
-like colonies. Bacterial species were identified by biochemical tests with an API 20E
identification kit (bioMérieux). E. coli O-serotyping, E. coli phylogenetic grouping and PFGE
typing were carried out as in our previous report [6]. MICs were determined via a Dry Plate
EIKEN (Eiken Chemical) and ETEST (bioMérieux) according to the manufacturers' instructions.
The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI)
guidelines [12]. Mutations in QRDR genes were confirmed by PCR amplification and sequencing
[13]. PMQR genes were screened by PCR [14–16]. The β-lactamase-producing phenotype and
β-lactamase genes were detected as in our previous report [6]. When several isolates were
cultured from the same animal, they were designated as the same clone if they exhibited the
same PFGE patterns. The correlation between the prevalence of fluoroquinolone-resistant E. coli
and individual attributes was estimated using Fisher’s exact test. A P value of <0.05 was
considered significant.

Go to:
Results
The correlation between individual animal attributes (sex, age group, presence/absence of an
owner, health status and enrofloxacin medication treatment) and the prevalence of
fluoroquinolone-resistant E. coli is presented in Table 1. Fluoroquinolone-resistant E. coli was
detected in 18 out of 38 dogs (47.4 %) and 14 out of 78 cats (17.9 %). Statistical analysis showed
that prevalence among dogs was significantly higher than among cats (P=0.0017), and prevalence
in males was significantly higher than in females among both dogs (P=0.0064) and cats
(P=0.0088). No significant correlations were found relating to the presence/absence of an owner
or treatment with enrofloxacin medication in the animal shelter.

Table 1.
Correlation between animal attributes and prevalence of fluoroquinolone-resistant E. coli .
Animal attributes

Detected/population (%)

Doga

18/38 (47.4 %)

Cata

14/78 (17.9 %)

Sex

Male

16/25 (64.0 %)b

11/36 (30.6 %)c

Female

2/13 (15.4 %)b

3/42 (7.1 %)c

Age group

Young

0/4 (0 %)

5/20 (25.0 %)

Mature to old

18/34 (52.9 %)

9/58 (15.5 %)

Ownerd

Presence
13/24 (54.2 %)

1/16 (6.3 %)

Absence

5/14 (35.7 %)

13/62 (21.0 %)

Health statuse

Healthy

15/32 (46.9 %)

11/59 (18.6 %)

Unhealthy

3/6 (50.0 %)

3/19 (15.8 %)

Enrofloxacinf

Medication

2/5 (40.0 %)

0/0 (0 %)

No medication

16/33 (48.5 %)

14/78 (17.9 %)

Open in a separate window

a, A significant difference (P=0.0017) was detected.

b, A significant difference (P=0.0064) was detected.

c, A significant difference (P=0.0088) was detected.

d, Presence or absence of owner when admitted to an animal shelter.

e, Unhealthy group included animals with emaciation, weakness, injury, symptoms of illness, or
malnutrition.

f, Enrofloxacin medication for treatment in an animal shelter.

The resistance mechanisms and characteristics of 32 fluoroquinolone-resistant E. coli isolates are

given in Table 2. All isolates showed MICs ≥256 µg ml−1 for nalidixic acid and demonstrated at
least one mutation in the QRDR. Four isolates with MICs of 2–3 µg ml−1 for ciprofloxacin
harboured a single mutation in gyrA. Fourteen isolates with MICs of 3–24 µg ml−1 for
ciprofloxacin carried two or three mutations in the QRDR: the combination of a double
mutation (except for isolate 37E-1) in gyrA and a single mutation in parC. Fourteen isolates with
MICs ≥32 µg ml−1 for ciprofloxacin carried a total of four mutations in the QRDR: the
combination of a double mutation in gyrA, a single mutation in parC and a single mutation in
parE; or the combination of a double mutation in gyrA and a double mutation in parC. For
PMQR, nine isolates harboured aac-(6′)-Ib-cr, five isolates harboured qnrS and one isolate
harboured qnrB. The qnrS gene was always harboured simultaneously with aac-(6 ′)-Ib-cr. A
relationship between the presence of PMQR and ciprofloxacin MIC values was not apparent.
There were seven isolates resistant to cefotaxime and six isolates produced β-lactamase from the
various β-lactamase gene(s). Extended-spectrum β-lactamase (ESBL) genes (CTX-M-14 or
CTX-M-27) were harboured simultaneously with aac-(6′)-Ib-cr, while the DHA-1 gene was
harboured with qnrB. For the O-serotype, most of the E. coli isolates were untyped, except for
serotypes O1, O25 and O157, each from one isolate. For phylogenetic grouping, three isolates
belonged to phylogenetic group B2, and 11 isolates belonged to group D, while the remaining
isolates belonged either to group A or to group B1.

Table 2.
Characteristics of fluoroquinolone-resistant E. coli isolates from dogs and cats

Isolate ID

Animal

species

MIC(μg ml−1)a for

QRDRb

PMQRc
O-serotype

Phylogenetic

group

MIC (μg ml−1)a for

β-Lactamase

(β-lactamase gene)

PFGE typed

Ciprofloxacin

Nalidixic acid

gyrA

parC

parE

Cefotaxime

Ser83

Asp87

Ser80

Glu84

Ser458

51–1

Dog

>256
Leu83

qnrS, aac-(6′)-Ib-cr

UT

<1

9′

64–2

Dog

256

Leu83

qnrS, aac-(6′)-Ib-cr

UT

D
<1

9′′′

72–1

Cat

>256

Leu83

qnrS, aac-(6′)-Ib-cr

UT

<1

9′′′′

63–1

Dog

>256

Leu83

－
－

qnrS, aac-(6′)-Ib-cr

UT

<1

9′′

32–1

Dog

>256

Leu83

Asn87

Ile80

UT

AmpC (CMY-2)

6
26–1

Cat

>256

Leu83

Tyr87

Ile80

O157e

B1

<1

10

89–1

Cat

>256

Leu83

Asn87

Ile80

－
UT

B1

<1

13

37E-1

Cat

>256

Leu83

Ile80

qnrS, aac-(6′)-Ib-cr

UT

<1

24–1

Dog

>256
Leu83

Asn87

Ile80

aac-(6′)-Ib-cr

UT

32

ESBL (CTX-M-14)

16

28–1

Cat

>256

Leu83

Asn87

Ile80

UT

B1
<1

12

88–1

Cat

>256

Leu83

Asn87

Ile80

UT

B1

<1

107–1

Dog

>256

Leu83

Asn87

Ile80
－

UT

B1

<1

108–1

Dog

>256

Leu83

Asn87

Ile80

UT

B1

<1

4′

57E-1

Cat

12
>256

Leu83

Asn87

Ile80

UT

B1

<1

11

40–1

Cat

16

>256

Leu83

Asn87

Ile80

UT

B1

<1
12′

2E-1

Cat

16

>256

Leu83

Asn87

Ile80

UT

B2

<1

73–1

Cat

16

>256

Leu83

Asn87

Ile80

－
－

UT

B2

<1

3′

13–1

Dog

24

>256

Leu83

Asn87

Ile80

qnrB

O1

32

AmpC (DHA-1)

7–1

Cat
32

>256

Leu83

Tyr87

Ile80

Ala458

aac-(6′)-Ib-cr

UT

16

ESBL (CTX-M-14, TEM-1)

42–1

Dog

>32

>256

Leu83

Asn87

Ile80

Ala458
UT

<1

85–1

Cat

>32

>256

Leu83

Tyr87

Ile80

Ala458

UT

>32

nd r

76–1

Dog

>32

>256
Leu83

Asn87

Ile80

Ala458

UT

B1

<1

14

83–1

Dog

>32

>256

Leu83

Asn87

Ile80

Ala458

UT

B1

<1

14
86–1

Dog

>32

>256

Leu83

Asn87

Ile80

Ala458

UT

B1

<1

14

96–1

Dog

>32

>256

Leu83

Asn87

Ile80

Ala458
UT

B1

<1

14

97–1

Dog

>32

>256

Leu83

Asn87

Ile80

Ala458

UT

B1

<1

14

98–1

Dog

>32

>256

Leu83
Asn87

Ile80

Ala458

UT

B1

<1

14

105–1

Dog

>32

>256

Leu83

Asn87

Ile80

Ala458

UT

B1

<1

14

106–1
Dog

>32

>256

Leu83

Asn87

Ile80

Ala458

UT

B1

<1

14

70–1

Dog

>32

>256

Leu83

Asn87

Ile80

Ala458

UT
B1

<1

14′

104–1

Cat

>32

>256

Leu83

Asn87

Ile80

Gly84

aac-(6′)-Ib-cr

UT

16

ESBL (CTX-M-14, TEM-1)

15

87–1

Cat

>32

>256
Leu83

Asn87

Ile80

Val84

aac-(6′)-Ib-cr

O25

B2

>32

ESBL (CTX-M-27)

17

Open in a separate window

a, MICs for ciprofloxacin, nalidixic acid and cefotaxim were confirmed with an E-test.

b, Mutation in gyrB was not found.

c, PMQR: qnrA [14], qnrB [14], qnrC [14], qnrD [16], qnrS [14], qepA [14], oqxA [15], oqxB [15] and
aac(6')-Ib-cr [14] were tested.

d, Prime symbols (', '', ''', '''' indicate >80 % similarity between band patterns.

e, Negative for the shiga toxin gene.

f, β-Lactamase phenotype and β-lactamase genes were not detected.

Antimicrobial susceptibility to nine antibiotics was tested (Fig. 1). The proportions that showed
resistance or intermediate resistance were as follows: 9.4 % for gentamicin, 0 % for
piperacillin/tazobactam, 0 % for meropenem, 21.9 % for cefazolin, 12.5 % for ceftazidime, 100 %
for ciprofloxacin, 40.6 % for trimethoprim-sulfamethoxazole, 68.8 % for ampicillin and 12.5 %
for amoxicillin-clavulanic acid.

An external file that holds a picture, illustration, etc.

Object name is acmi-2-077-g001.jpg
Fig. 1.
PFGE analysis and antimicrobial susceptibility. Xba I-digest PFGE patterns and a dendrogram
based on band pattern similarity are shown. For PFGE type, similarity <80 % is indicated as a
different number, and ≥80 % is indicated as the same number with prime symbols ( ′, ′′, ′′′, ′′′′).
Susceptibility to gentamicin (GM), piperacillin/tazobactam (TAZ/PIPC), meropenem (MEM),
cefazolin (CEZ), ceftazidime (CAZ), ciprofloxacin (CPFX), trimethoprim-sulfamethoxazole (ST),
ampicillin (ABPC) and amoxicillin-clavulanic acid (ACV) is indicated by shaded boxes. Black
boxes, resistant; grey boxes, intermediate; white boxes, susceptible.

PFGE and a dendrogram analysis were performed to assess genetic relationships (Fig. 1). There
were 17 distinct patterns with <80 % similarity from the 32 fluoroquinolone-resistant E. coli
isolates. The similarity of band patterns ranged from 14.3 to 100 %. There were five PFGE types
that indicated >80 % similarity, for a total of 20 isolates (Table 2, Fig. 1).

Go to:
Discussion
In this study, 47.4 % of dogs and 17.9 % of cats carried fluoroquinolone-resistant E. coli . The
reason for the significant difference in prevalence rates between dogs and cats, or between males
and females, was not clear; however, isolates showing the same PFGE types were detected in
multiple dogs, and this may have influenced the prevalence rates. Additionally, the presence of a
previous owner was not necessarily related to possessing the bacteria. Sato et al. found that a
high prevalence of fluoroquinolone-resistant E. coli may have been caused by frequent or
continuous fluoroquinolone use in animal hospitals [9]. In the present study, the use of
enrofloxacin in the animal shelter was not related to possessing the bacteria, although a full
medication history was not determined.

Co-resistance to cephalosporin was observed in 21.9 % (7/32) of fluoroquinolone-resistant

isolates. These included four ESBL-producing and two AmpC-producing isolates. Moreover,
68.8 % (22/32) of fluoroquinolone-resistant isolates were also resistant to other categories of
antimicrobials. Fluoroquinolone-resistant E. coli with co-resistance to other antimicrobials,
especially cephalosporin, is prevalent nationwide [3, 4], and has also been detected among
clinical E. coli isolated from companion animals [10, 17]. According to the international standard
definition for multidrug-resistant (MDR) bacteria [18], 15 isolates (46.9 %) were classified as
MDR with non-susceptibility to three or more antimicrobial categories. These findings may have
great relevance for the treatment of infectious diseases in both animals and humans.

For QRDR, all isolates harboured a mutation in gyrA at Ser83, the most common mutation site.
Highly resistant isolates carried a combination of mutations within gyrA and parC, and the
number of mutations was proportional to the MIC for ciprofloxacin. In line with previous reports
regarding fluoroquinolone-resistant E. coli from companion animals [7, 10], mutations in QRDR
genes play a significant role in mediating resistance to fluoroquinolone. For PMQR genes on
transferable plasmids, which confer a lower level of resistance to fluoroquinolone than QRDR [4,
5], aac-(6′)-Ib-cr was the most prevalent, followed by qnrS and qnrB. The predominance of the
aac-(6′)-Ib-cr gene and qnr variant genes agrees with the previous reports of E. coli isolates
recovered from companion animals in the USA [10] and Europe [7, 8]. This trend has been
confirmed in human clinical isolates as well [3]. Furthermore, many different PMQR genes have
also been found in E. coli isolates from Chinese companion animals [11].

PFGE patterns were widely diverse, but some isolates showed the same or similar band patterns.
Although these animals were from separate locations, cross-contamination may have occurred
on admittance to the animal shelter or may also have been caused by the movement of staff or
materials.

In conclusion, we found that fluoroquinolone-resistant E. coli was widely distributed among

sheltered companion animals with a wide range of individual attributes. This may be related to
the dissemination of fluoroquinolone resistance among Japanese human clinical E. coli [2],
livestock animals [19] and different environments [20]. To lower the overall fluoroquinolone
resistance, it will be necessary to reduce fluoroquinolone use and continue surveillance not only
in the human clinical field but also in the veterinary field.

Consent for publication

This paper does not include details, images or videos relating to an individual person. Therefore,
informed consent is not necessary.

Go to:
Data bibliography
This paper does not include data reference.

Go to:
Funding information
This work was partially supported by JSPS KAKENHI, Grant Number 16H07501.

Go to:
Acknowledgements
We thank the Osaka City Health Service Bureau for the organization of this work.

Go to:
Author contributions
K. U., carried out conceptualization, investigation, formal analysis and writing. H. A., and A. F.,
contributed to methodology and investigation. M. M., and T. H., contributed to investigation
and resources. J. O., contributed to the supervision of this work.

Go to:
Conflicts of interest
The authors declare that there are no conflicts of interest.

Go to:
Ethical statement
The authors conformed to Animal Research: Reporting of In Vivo Experiments guidelines. This
research did not contain experimental work with humans, and it was approved that additional
ethical review or permission was unnecessary by the ethical review committee of Osaka Institute
of Public Health, according to the ‘ethical guidelines for medical and health research involving
human subjects’ established by the Ministry of Health, Labor and Welfare in Japan.

Go to:
Footnotes
Abbreviations: ESBL, extended-spectrum β-lactamase; MDR, multi-drug resistance; PMQR,
plasmid-mediated quinolone resistance; QRDR, quinolone resistance-determining region.

Repositories - This paper contains no new sequence data.

Go to:
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Journal ListAccess Microbiolv.2(1); 2020PMC7525059

Logo of accessmicro
Access Microbiol. 2020; 2(1): acmi000074.
Published online 2019 Oct 31. doi: 10.1099/acmi.0.000074
PMCID: PMC7525059
PMID: 33062933
The tough process of unmasking the slow-growing mycobacterium: case report of
Mycobacterium microti infection
Cornelia A. M. van de Weg, 1 ,* Jurriaan E. M. de Steenwinkel, 1 Jelle R. Miedema, 2 Marleen
Bakker, 2 Jakko van Ingen, 3 and Wouter Hoefsloot 3 , 4
Author information Article notes Copyright and License information Disclaimer
Go to:
Abstract
Mycobacterium microti belongs to the Mycobacterium tuberculosis complex (MTBC). It can
cause pulmonary and extrapulmonary tuberculosis in humans. Compared to M. tuberculosis ,
which is the most prevalent subspecies of the MTBC, M. microti infection has a different
etiology. Moreover, establishing the diagnosis with conventional bacteriology can be difficult.
We will illustrate this with a case of an extrapulmonary tuberculosis of the hip caused by M
.microti in an immunocompetent patient in The Netherlands.

Keywords: Mycobacterium microti, mycobacterium tuberculosis complex, extrapulmonary

tuberculosis
Go to:
Case report
A 45-year-old man presented at the emergency department of our tertiary teaching hospital with
a septic arthritis of the left hip. Four weeks before presentation the hip became painful. Since
then the pain worsened and the patient became febrile. He did not suffer from night sweats.

The patient had no significant medical history. He originated from former Yugoslavia, where he
spent the holidays once or twice a year. His occupation was supervisor in a metal recycling
company in the harbour of Rotterdam.

On physical examination the hip was mildly swollen and painful at palpation. Laboratory
investigation showed an increased C reactive protein (CRP) of 234 mg l−1 (normal <10) with a
normal leukocyte count. Magnetic resonance imaging (MRI) of the hip was performed, which
showed a high-intensity signal and a lot of intra-articular fluid at the swollen side of the hip.
With an ultrasound guided biopsy synovial fluid was aspirated and sent to the microbiology
department.

The working hypothesis was an ordinary septic arthritis. However, a gram stain of the synovial
fluid did not show any micro-organisms. Antibiotic treatment was started immediately with
intravenous cefuroxime and one dose of gentamicin, according to local antibiotic guidelines.
After starting antibiotic treatment the fever and increased CRP persisted. Therefore, a chest
radiograph was taken, which showed bilateral, perihilar infiltrates consisting of several small
nodules. A chest computed tomography (CT) showed perihilar diffuse nodular infiltrates (Fig. 1).

An external file that holds a picture, illustration, etc.

Object name is acmi-2-074-g001.jpg
Fig. 1.
Chest computed tomography, showing perihilar consolidations at both sides with underlying
bronchiectasis. In the periphery of the lungs smaller nodular infiltrates are visible. These are
indicated by the arrows.
With a differential diagnosis of pulmonary and extrapulmonary tuberculosis, the hip synovial
fluid was stained with auramine-rhodamine and then acid-fast bacilli could be visualized. Next,
GeneXpert MTB/Rif (Cepheid, Sunnyvale, CA, USA), a molecular test based on nucleic acid
amplification, confirmed M. tuberculosis complex in the sample from the synovial fluid. The
normal aerobic and anaerobic cultures were negative. The mycobacterial culture, using our BD
BACTEC MGIT 960 mycobacteria culture system (BD Diagnostics, Sparks, MD, USA) was
negative after 6 weeks of culture. Given the positive GeneXpert and no history of pre-treatment,
it was decided to extend the culture period for another 6 weeks. After a total of 75 days of
culture, the mycobacterial culture became positive (Fig. 2). In order to differentiate within the
M. tuberculosis complex we used the GenoType MTBC molecular genetic assay (HAIN
Lifescience, Nehren, Germany) and identified the strain as an M. microti . Additionally, the
GenoType MTBDRplus (HAIN Lifescience) was performed in order to identify molecular genetic
resistance towards isoniazid (INH) and rifampicin (RMP). This hybridization assay showed no
mutations in the rpoB gene (associated with RMP susceptibility) and no mutations in the katG
or inhA genes (associated with INH susceptibility).

An external file that holds a picture, illustration, etc.

Object name is acmi-2-074-g002.jpg
Open in a separate window
Fig. 2.
Microscopy after kinyoun staining of M. microti in culture. This culture became positive after
75 days of incubation. The microscope used was a Zeiss Axioskop, the magnification ×100.

The sputum of the patient was also tested for mycobacteria, but turned out to be negative by
auramine-rhodamine staining, GeneXpert and (mycobacterial) culture. Eventually,
broncho-alveolar lavage was performed, but with the previous three tests M. microti could not
be detected.

The M. microti infection was treated with INH, RMP, pyrazinamide and ethambutol according to
the World Health Organization recommended dosage [1]. Initially the patient recovered and the
fever and pain in the hip disappeared. Therefore, the pyrazinamide and ethambutol were
discontinued after 2 months. However, shortly thereafter the former complaints reoccurred and
the pyrazinamide and ethambutol were restarted. A CT scan of the hip revealed bone
destruction at the anterior side and multiple abscesses in the adjacent muscles (Fig. 3). A
radiological drainage of these abscesses was performed. Therapeutic drug monitoring of RMP
and INH showed that drug levels of both drugs were subtherapeutic. Hereafter, the dose of INH
was increased to 900 mg per day and RMP to 1800 mg per day. Shortly after this treatment
modification the patient got complaints of polyneuropathy. Therefore, the INH was
discontinued. The other drugs were continued and eventually, the patient improved clinically.

An external file that holds a picture, illustration, etc.

Object name is acmi-2-074-g003.jpg
Open in a separate window
Fig. 3.
Computed tomography of the left hip, showing bone destruction of the anterior side of the
collum. Next to this site, multiple fluid collections are visible, which are highly suspicious for
abscesses. These collections infiltrate in the adjacent muscles. The largest collection in this view
measures 64.5 by 37.1 mm and has a very thick wall.

Go to:
Discussion
This case report presents a patient with an unexpected diagnosis of M. microti tuberculosis.

M. microti is part of the MTBC complex, which among others include M. tuberculosis, M. bovis,
M. africanum, ‘M. canettii’, M. pinnipedii and M. caprae .

Studies conducted in France and Great Britain showed that M. microti is endemic in small
rodents, such as mice and voles, who serve as the reservoirs [2, 3]. Infections of other animals,
such as pigs, ferrets, cats and dogs, have also been described. Until 1998 it was believed that M.
microti exclusively caused disease in animals, but then the first four cases were described in
humans [4]. Since then, M. microti is considered a zoonotic pathogen, which can infect humans
through spillover hosts, such as dogs or cats or through faecal contamination of the
environment. One case series suggests that human-to-human transmission may have occurred
[4]. For our patient the source of infection was not clear.

In 2010, only 27 human cases of M. microti had been described in the literature [5]. It is believed
that the prevalence of infections with M. microti is underestimated. One reason is that it is easily
missed in routine bacteriology. M. microti is extremely slow growing and to culture it, it often
takes more than 6–8 weeks. M. microti can be distinguished from M. tuberculosis by the typical
curved morphology of the bacteria at Ziehl–Neelsen staining. However, given the subtle
differences, this might be missed and regarded M. tuberculosis if no additional genotypically
testing is performed. Therefore, nowadays, molecular assays, such as the GenoType MTBC, have
become part of routine laboratory diagnostics in high- and middle-income countries. These
assays can distinguish between the members of the MTBC. Moreover, they can be used on direct
patient material instead of cultured strains if the mycobacterial load is high enough, which
speeds up the time to diagnosis [2, 6].

It is expected that with increased use of molecular techniques in diagnostic procedures, more
infections with M . microti will be discovered. In a recent study, conducted in South Africa,
genotyping of 215 M . tuberculosis isolates showed that 1.9 % were M. microti [7].

M. microti can cause disease independently of the immune status of the patient. In a report from
Panteix et al. from the 27 cases, ten were immunocompetent [5]. From the other 17 cases, the
immunological status was compromised by HIV infection, kidney transplantation or diabetes.
Our patient proved HIV negative and had no other signs or test results indicative of a
compromised immunological status.

In the majority of cases M. microti causes pulmonary tuberculosis, but extrapulmonary

tuberculosis has also been described. Of the cases with extrapulmonary tuberculosis two
patients had an abdominal infection and one an osteomyelitis of the hip [8]. Our case would be
the fourth case of extrapulmonary M. microti infection ever published.

The empirical treatment chosen in this patient, is based on the fact that M. microti usually
responds well to conventional treatment. However, in the case series from Panteix et al.
resistance to pyrazinamide was reported in one of the six cases described [5].

In conclusion, we summarized a patient who was diagnosed after a significant time delay with M.
microti tuberculosis. Establishing the diagnosis with conventional bacteriology can be difficult
because this mycobacterium takes longer to grow in culture than M. tuberculosis . Molecular
assays can identify M .microti subspecies and decrease diagnostic delay.

Go to:
Funding information
This work received no specific grant from any funding agency.

Go to:
Acknowledgements
Authors would like to thank the patient, who was willing to share his medical history in this case
report, and A. Ophorst and J. den Boer for their photograph of the ZN-stained M. microti.

Go to:
Conflicts of interest
The authors declare that there are no conflicts of interest.

Go to:
Ethical statement
The patient, described in this case report, has read the manuscript and signed for consent to
publish. The corresponding author has obtained written informed consent from the patient
before submission.

Go to:
Footnotes
Abbreviations: CRP, C reactive protein; CT, Computed Tomography; INH, Isoniazid; MRI,
Magnetic Resonance Imaging; MTBC, Mycobacterium Tuberculosis Complex; RMP, Rifampicin.

Go to:
References
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