An insight into selection specificity of quinolone resistance determinants within Enterobacteriaceae family

Accepted Manuscript Title: An insight into selection specificity of quinolone resistance determinants within Enterobacteriaceae family Authors: Nivedita Dasgupta, Deepjyoti Paul, Debadatta DharChanda, Birson Ingti, Dibyojyoti Bhattacharjee, Atanu Chakravarty, Amitabha Bhattacharjee PII: DOI: Reference:

S2213-7165(17)30075-9 http://dx.doi.org/doi:10.1016/j.jgar.2017.03.010 JGAR 400

To appear in: Received date: Revised date: Accepted date:

28-5-2016 17-1-2017 8-3-2017

Please cite this article as: Nivedita Dasgupta, Deepjyoti Paul, Debadatta DharChanda, Birson Ingti, Dibyojyoti Bhattacharjee, Atanu Chakravarty, Amitabha Bhattacharjee, An insight into selection specificity of quinolone resistance determinants within Enterobacteriaceae family (2010), http://dx.doi.org/10.1016/j.jgar.2017.03.010 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.

An insight into selection specificity of quinolone resistance determinants within Enterobacteriaceae family

Nivedita Dasgupta a, Deepjyoti Paul a, Debadatta Dhar(Chanda) b, Birson Ingti a, Dibyojyoti Bhattacharjee c, Atanu Chakravarty b, Amitabha Bhattacharjee a,*

a

Department of Microbiology, Assam University, Silchar, Assam, India

b

Department of Microbiology, Silchar Medical College and Hospital, Silchar, Assam,

India c

Department of Statistics, Assam University, Silchar, Assam, India

* Corresponding author. Tel.: +91 84 7193 9088. E-mail address: [email protected] (A. Bhattacharjee).

ARTICLE INFO Article history: Received 28 May 2016 Accepted 8 March 2017

Highlights 

Inappropriate use of quinolones has led to the development of resistance to this group of antimicrobials.



Study of the carriage of plasmid-mediated quinolone resistance (PMQR) genes and dissemination among Enterobacteriaceae.



Insight into plasmid selection due to quinolone stress and their role in horizontal expansion.



Selection and maintenance of quinolone resistance genes showed bias towards specific groups of quinolones.



Specificity/bias can be used to develop a phenotypic marker for detection of PMQR genes.

ABSTRACT Objectives: Quinolone antimicrobials are frequently misused due to self-medication and suboptimal dose administration, leading to the development of resistance as well as treatment failure. The present study aimed to characterise plasmid-mediated quinolone resistance (PMQR) determinants and their genetic selection in the presence of quinolone stress within members of the Enterobacteriaceae. Methods: A total of 209 non-duplicate Enterobacteriaceae isolates were collected from hospital and community health centres over the period July 2013 to June 2014. Molecular characterisation of phenotypically screened quinolone-resistant isolates was done by multiplex PCR. Plasmids bearing the qnr and aac(6′)-Ib-cr genes were transformed into Escherichia coli DH5 and were selected on Muller–Hinton agar plates containing 0.25 g/mL and 0.5 g/mL ciprofloxacin, norfloxacin, ofloxacin, levofloxacin and moxifloxacin. Conjugation experiments were performed to

determine whether the aac(6′)-Ib-cr- and qnr-carrying plasmids were selftransferable. Results: The transformation assay revealed that transformants carrying qnrA could be selected in media containing norfloxacin, ciprofloxacin and levofloxacin, whereas qnrB and aac(6′)-Ib-cr were selected on media containing norfloxacin and ciprofloxacin. Transformed qnrD could be selected in media containing norfloxacin and ofloxacin, and qnrS was selected only in the presence of levofloxacin. Conclusions: The presence of qnr genes has been associated with an increase in quinolone minimum inhibitory concentrations (MICs) and therefore leads to treatment failure when quinolones are used as selective therapeutic drugs. Since PMQR determinants have a high prevalence, effective measures should be taken and surveillance should be performed in order to avoid treatment failures using this group of antimicrobials.

Keywords:Antimicrobial use Resistance gene Plasmid-mediated antimicrobial resistance Quinolone resistance

1. Introduction Plasmid-mediated quinolone resistance (PMQR) among Enterobacteriaceae has been reported from different parts of the world since the last decade. Qnr-like proteins belonging to the pentapeptide repeat family protect DNA gyrase from quinolone binding to type II topoisomerases. To date, five qnr determinants [qnrA (six variants), qnrB (six variants), qnrC, qnrD, qnrS (two variants)] as well as aac(6′)Ib-cr have been identified worldwide [1]. Among them, the qnrB determinant is

associated with increased resistance to fluoroquinolones. Appropriate antimicrobial usage in the management of infectious diseases is an important factor regarding the development of antimicrobial resistance. The issue is particularly relevant in many low- and middle-income countries where the sale of antimicrobials is often unrestricted [2–4]. Selection of resistant bacteria is encouraged by the sustained usage of some antimicrobial classes over a period of time, which reduces the effect of these drugs on pathogens resulting in a resistant phenotype [5–7], consequently leading to treatment failure, increased treatment costs and prolonged therapy [8]. Therefore, it is of interest to know whether these quinolone resistance determinants are selected within the host irrespective of quinolone pressure or whether they exhibit specificity in selection towards particular quinolone drugs. Thus, the current study aimed to characterise PMQR determinants and their genetic selection in the presence of quinolone stress.

2. Materials and methods 2.1. Bacterial isolates Consecutive non-duplicate isolates of Enterobacteriaceae were collected from community health centres in and around Silchar town (Assam, India) and from patients who had attended the clinics or who were admitted to different wards of Silchar Medical College and Hospital (SMCH) (Silchar, Assam, India) from July 2013 to June 2014. Patients were treated for complications such as urinary tract infection, post-surgical wound infection, gastrointestinal tract infection, and skin and soft-tissue infection. Isolates were collected from urine, stool, pus, sputum and blood samples. Isolates were identified based on conventional biochemical tests such as catalase,

oxidase and indole production tests, methyl red test, Voges–Proskauer test, citrate utilisation, urease production, sugar fermentation, phenyl alanine deaminase test, amino acid decarboxylase and dihydrolase test, oxidative fermentation test, nitrate reduction test and triple sugar iron test, as well as culture characteristics.

2.2. Screening of quinolone resistance Quinolone resistance was determined by the disk diffusion method using nalidixic acid (30 g), norfloxacin (10 g), ciprofloxacin (5 g), ofloxacin (5 g), lomefloxacin (5 g), gatifloxacin (5 g), gemifloxacin (5 g), sparfloxacin (5 g), levofloxacin (5 g) and moxifloxacin (5 g) (HiMedia, Mumbai, India). Escherichia coli ATCC 25922 was used as the quality control strain for antimicrobial susceptibility testing and the results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines [9].

2.3. Characterisation of quinolone resistance by multiplex PCR DNA extraction was performed using an improved boiling centrifugation method for quinolone-non-susceptible isolates [10]. The presence of qnrA, qnrB, qnrS, qnrD, qnrC and aac(6′)-Ib-cr genes was detected by a PCR-based technique using the primers shown in Supplementary Table S1. Each single reaction mixture (25 L) contained 1 L (10 ng) of DNA, 15 pmol of each primer, 12.5 L of 2 GoTaq® Green (Promega, Madison, WI) and nuclease-free water. Previously confirmed isolates harbouring qnrA, qnrB, qnrS, qnrD, qnrC and aac(6′)-Ib-cr were used as positive controls, and E. coli ATCC 25922 was used as a negative control. Reactions were run under the following conditions: initial denaturation at 95 C for 2 min; 35 cycles of

95 C for 50 s, 53 C for 40 s and 72 C for 1.20 min; and a final extension at 72 C for 5 min. PCR products were sequenced and analysed using the blast tool of the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.gov/BLAST).

2.4. Plasmid analysis, transformation and conjugation assays Plasmid DNA was extracted using a QIAGEN Mini Prep Kit (QIAGEN, Hilden, Germany) and was transferred into E. coli DH5 by the heat shock method [11]. Transformants were selected on Luria–Bertani agar plates (HiMedia) containing 0.25 g/mL and 0.5 g/mL of norfloxacin, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin each. Transformants were screened for their plasmid content and resistance phenotype. The experiment was repeated three times. To investigate the horizontal transferability of plasmids encoding quinolone resistance, conjugation was performed using a streptomycin-resistant E. coli strain B (Genei, Bangalore, India) as recipient and mating was performed as described previously [12].

2.5. Plasmid incompatibility typing Plasmid incompatibility was determined for transformants and transconjugants harbouring qnr and aac(6′)-Ib-cr genes by the PCR-based replicon typing method [13].

2.6. Antibiotic susceptibility and minimum inhibitory concentration (MIC) determination Susceptibility was determined by the disk diffusion method for the following antimicrobials: ampicillin (10 g); cefotaxime (30 g); ceftriaxone (30 g); ceftazidime (30 g); imipenem (10 g); trimethoprim/sulfamethoxazole (25 g); tigecycline (15 g); gentamicin (120 g); amikacin (30 g); and polymyxin B (300 U) (HiMedia).

In addition, the MICs of ciprofloxacin, norfloxacin (Cipla Ltd., Mumbai, India), ofloxacin (Micro Labs Ltd., Bangalore, India), levofloxacin (Ajanta Pharma Ltd., Mumbai, India) and moxifloxacin (Sun Pharmaceuticals Ltd., Mumbai, India) were determined by the agar dilution method. Escherichia coli ATCC 25922 was used as the quality control strain. Results were interpreted according to CLSI guidelines [9], except for tigecycline which were interpreted according to US Food and Drug Administration (FDA) breakpoints [14].

2.7. Typing of isolates All qnr-positive isolates were typed by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Each single reaction mixture (25 L) contained 1 L (10 ng) of DNA suspension, 15 pmol of each primer, 12.5 L of 2 GoTaq® Green Master Mix (Promega) and nuclease-free water. Primers used and reaction conditions were similar to those described previously [15].

3. Results Of 512 consecutive non-duplicate isolates, 209 (40.8%) were identified as members of the Enterobacteriaceae family, from community health centres (n = 115) and SMCH (n = 94). The isolates included E. coli (n = 139), Klebsiella pneumoniae (n = 53), Klebsiella oxytoca (n = 7), Proteus vulgaris (n = 8) and Proteus mirabilis (n = 2). Of the 209 Enterobacteriaceae isolates, 183 were screened phenotypically as quinolone-resistant, showing non-susceptibility to at least one of the tested quinolone group of drugs tested (Supplementary Table S2). Resistance to nalidixic acid was most common (91.3%), followed by ciprofloxacin (78.1%), norfloxacin (72.1%) and ofloxacin (67.2%) (Supplementary Table S3). The resistance patterns of the isolates towards quinolone antibiotics are shown in Table 1.

A total of 93 isolates showed amplification in the multiplex PCR, of which 59 E. coli (46.8% of the 126 quinolone-resistant E. coli), 26 K. pneumoniae (57.8% of the 45 quinolone-resistant K. pneumoniae), 2 K. oxytoca (66.7% of the 3 quinoloneresistant K. oxytoca), 5 P. vulgaris (71.4% of the 7 quinolone-resistant P. vulgaris) and 1 P. mirabilis (50.0% of the 2 quinolone-resistant P. mirabilis) were positive for PMQR determinants. The prevalence of qnrD was highest amongst the qnr genes (Table 1; Figs 1–3); none of the isolates showed the presence of qnrC. Sequencing results established the presence of variants such as qnrA1, qnrS1 and qnrB7 in the isolates. All of the gene types were successfully transferred into E. coli strain DH5 by transformation. In the transformation assay it was seen that transformants carrying qnrA could be selected in media containing norfloxacin, ciprofloxacin and levofloxacin, whereas qnrB and aac(6′)-Ib-cr were selected on media containing norfloxacin and ciprofloxacin. Transformed qnrD could be selected in media

containing norfloxacin and ofloxacin, and qnrS was selected only in the presence of levofloxacin. Horizontal transfers of all of the qnr genes was successful in conjugation assays. However, medium containing moxifloxacin was unable to select any of the qnr determinants or aac(6′)-Ib-cr. The analysed plasmid-encoded qnr and aac(6′)-Ib-cr genes belonged to IncP, HI2, FIIs, K/B, F, Y, P, FIC, T, A/C and FrepB replicon types. DNA fingerprinting by ERIC-PCR showed that 58 haplotypes existed in E. coli, 37 in K. pneumoniae and 3 in P. vulgaris (Fig. 4). Polymyxin B and other antimicrobials such as imipenem and cephalosporins showed good antimicrobial activity (Table 2). The MIC results showed that all parent isolates had high MICs and that transformants had MICs above the breakpoint for norfloxacin, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin (Table 3).

4. Discussion The widespread dissemination of quinolone resistance determinants and aac(6′)-Ibcr could enhance the rapid development of fluoroquinolone resistance. The enterobacterial isolates used in this study showed specificity in resistance or decreased susceptibility to different quinolone antibiotics. The present study has demonstrated decreased quinolone and fluoroquinolone susceptibility as well as resistance to these agents.

This study clearly indicated the presence of fluoroquinolone-resistant enterobacterial strains both in hospital and community settings. The prevalence rates of quinolone resistance within E. coli, K. pneumoniae and P. vulgaris were 90.6% (126/139), 84.9% (45/53) and 87.5% (7/8) respectively. Compared with these results, a previous study reported a different pattern in Tunisia [16], where resistance rates

were 30.24% for E. coli, 46.26% for K. pneumoniae and 2.13% for P. vulgaris. A recent study conducted in Algeria has also reported reduced susceptibility of uropathogenic E. coli towards nalidixic acid [17].

The remarkable prevalence of qnrD (23.5%) and qnrS (2.7%) discovered in 183 enterobacteria in the current study is quite distinct to that reported from Italy [18] and Argentina [19]. In the latter study, the prevalence of qnrD (2.5%; 2/81) in human clinical isolates from patients admitted to hospitals was reported but the sample set used only the tribe Proteeae (Proteus spp., Morganella morganii and Providencia stuartii). However, a similar study carried out in Brazil among community-acquired enterobacterial isolates showed a low prevalence of quinolone resistance determinants [20]. In the current study, PMQR genes accounted for 50.8% (93/183) of phenotypic resistance, among which the most prevalent genes were qnrD and aac(6′)-Ib-cr. Whereas earlier studies in India reported 67% and 54% PMQR in clinical isolates of K. pneumoniae and other enterobacterial isolates from environmental sources, respectively [21–23]. However, in other findings the aac(6′)Ib-cr gene accounted for 90% of the PMQR genes from hospital waste water [24]. This high incidence of qnrD and aac(6′)-Ib-cr is an alarming situation, indicating the spread of these genes in other species of the large Enterobacteriaceae family. The current study has mainly been done with a view to determine the existence of any selection specificity of quinolone resistance determinants. All of the aac(6′)-Ib-cr and qnr determinants identified in this study were transformed and the transformants were selected against ciprofloxacin, ofloxacin, norfloxacin, levofloxacin and moxifloxacin. Ciprofloxacin, which is a member of the large and widely used fluoroquinolone group of antimicrobial drugs, is considered as the empirical choice of

treatment for infections in adults. However, the extensive misuse and self-medication of these drugs, namely norfloxacin and ciprofloxacin, in the last two decades has led to the development of resistance within pathogens associated with communityacquired infections. Misuse of antibiotics through over-the-counter dispensing in India was supported by a study conducted in West Bengal [25]. Furthermore, studies by Patel et al. [26] and Chaudhary et al. [27] provide evidence in support of selfmedication leading to the development of resistance against fluoroquinolones and other groups of antimicrobials. Resistance against these quinolone drugs is associated with either the presence of qnr or mutation in the quinolone resistancedetermining region (QRDR) or maybe some other co-existing mechanism. This study has established a scenario where it has been observed that the presence of qnrA, qnrB, qnrD and aac(6′)-Ib-cr is associated with resistance to norfloxacin and ciprofloxacin. This study could give an insight into the usage pattern of different quinolone antimicrobials and their role in the carriage of specific quinolone resistance determinants within bacterial isolates, although other co-existing genes may be encoded within the same plasmid. The predominance of different quinolone resistance determinants [qnrD, aac(6′)-Ib-cr, qnrA, qnrB, qnrS] and their selection against corresponding quinolone groups corroborates well with the maximal usage of these antimicrobials in community-acquired infections and their over-the-counter availability. All of the qnrS (n = 5) plasmids were selected only in the presence of levofloxacin, and resistance against this drug can be predicted as a phenotypic marker for carriage of qnrS. However, use of levofloxacin is quite low compared with other fluoroquinolones and is possibly the reason behind the lower occurrence of this particular gene in the present study.

In accordance with a previous study conducted in Turkey [12], qnrA shows resistance against ciprofloxacin and nalidixic acid, which is in agreement with the fact that high consumption of fluoroquinolones and antimicrobial burden may affect the prevalence of quinolone resistance and may provide conditions for the selection of qnrA determinants. Selection of qnrA determinants could be also due to the presence of a successful plasmid carrying many other resistance determinants.

ERIC-PCR refers to the general method that utilises oligonucleotide primers matching palindromic repeated sequences described in enterobacteria to yield DNA fingerprints of individual bacterial isolates. Genetic divergence and homogeneity are apparent from the phylogenetic tree. DNA fingerprinting of all quinolone-resistant isolates by ERIC-PCR showed 58 haplotypes for E. coli, 37 for K. pneumoniae and 3 for P. vulgaris in this study, indicative of clonal diversity in the study area.

5. Conclusions This study stresses an urgent need for review of fluoroquinolone therapy and their prophylactic use both in community- and hospital-acquired infections. More molecular epidemiological investigations must be carried out to correlate the maintenance and persistence of resistance genes within a host when quinolone treatment is initiated. Thus, the study presents a scenario of plasmid selection in pathogens owing to quinolone stress or possibly due to other co-existing resistance determinants and other antibiotic pressure that might play a key role in plasmid selection and horizontal expansion.

Funding: None.

Ethical approval: This work was approved by the Institutional Ethical Committee of Assam University [no. IEC/AUS/C/2014-002dt-14/08/14].

Competing interests: None declared.

Acknowledgments: The authors would like to acknowledge the help of the Head of Department of the Department of Microbiology (Assam University, Silchar, Assam, India) for providing the infrastructure. The authors also acknowledge help from Assam University Biotech Hub for providing laboratory facilities to complete this work.

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Fig. 1. Amplicons of the first multiplex PCR. Lane L, 100 bp ladder; lane1, qnrS (positive control); lane2, qnrS; lane 3, aac(6′)-Ib-cr (positive control); lane 4, aac(6′)Ib-cr.

Fig. 2. Amplicons of the second multiplex PCR. (A) Lane L, size ladder; lane 1, qnrD (positive control); lanes 2–4, qnrD; (B) lane L, 100 bp ladder; lane 1, qnrA (positive control); lane 2–4 and 8, qnrA (628 bp); lane 12, qnrB (positive control; lanes 12–14 and 16, qnrB (546 bp).

(A)

(B)

Fig 3. Amplicons of the simplex PCR showing amplification of qnrC. Lane L, 100 bp ladder; lanes 1–3, test sample; lane 4, positive control of qnrC (447 bp).

Fig. 4. Results of ERIC-PCR fingerprinting: dendrogram to illustrate the clonal differences of (A) quinolone-resistant Escherichia coli and (B) quinolone-resistant Klebsiella pneumoniae.

1.00

1.29

1.58

1.87

2.16

Co-efficient

(A)

1.00

1.45

1.90

Co-efficient

(B)

2.34

2.79

Table 1 Quinolone resistance patterns of 183 quinolone-resistant Enterobacteriaceae isolates No. of antimicrobials resistant to and

No. of

aac(6′)-Ib-cr and

Type of Inc

pattern

strains

qnr determinants

plasmid

NAL

9

n/a (9)

FrepB

NOR

2

n/a (2)

K/B, F

LOM

1

n/a (1)

P

NAL/LOM

7

n/a (7)

FrepB, Y, P

CIP/LOM

4

n/a (4)

P

SPX/LOM

4

n/a (4)

P, A/C

NAL/LOM/GEM

4

n/a (4)

HI2, FIIs

NAL/CIP/LOM

2

n/a (2)

FrepB

NAL/NOR/LOM

17

qnrB (4)

FIIs, K/B

qnrD (1)

K/B, FrepB

n/a (12)

P, HI2

qnrD (2)

FrepB

One antimicrobial

Two antimicrobials

Three antimicrobials

Four antimicrobials NAL/CIP/NOR/GEM

2

NAL/CIP/LOM/GEM

1

n/a (1)

FrepB

17

qnrA (4)

P, K/B, HI2,

Five antimicrobials NAL/CIP/SPX/LOM/GEM

NAL/OFX/SPX/LOM/GEM

11

n/a (13)

A/C

qnrD (8)

FrepB

n/a (3)

HI2, FIIs FrepB, P

Six antimicrobials NAL/CIP/OFX/SPX/LOM/GEM

NAL/CIP/NOR/SPX/LOM/GEM

13

11

qnrA, qnrD (1)

K/B

qnrA (3)

P, K/B, FrepB

n/a (9)

K/B, FrepB

qnrA, qnrD (2)

HI2, FIIs

qnrB, aac(6′)-Ib-

FrepB, K/B

cr (2)

FrepB

n/a (7) Seven antimicrobials NAL/CIP/NOR/OFX/SPX/LVX/GEM

78

qnrA (4)

FrepB

qnrB (2)

P

qnrS (5)

K/B, FrepB

qnrD (21)

P, K/B,

aac(6′)-Ib-cr (23)

FrepB, T,

qnrA, qnrB (2)

A/C

qnrA, aac(6′)-Ibcr (1) qnrD, aac(6′)-Ibcr (8) n/a (12)

FIIs, K/B, F, Y, P, FIC, T, A/C K/B, P K/B Y,P,FIC,T FrepB

NAL, nalidixic acid; NOR, norfloxacin; LOM, lomefloxacin; CIP, ciprofloxacin; SPX, sparfloxacin; GEM, gemifloxacin; OFX, ofloxacin; LVX, levofloxacin; n/a, not amplified.

Table 2 Antimicrobial susceptibility of quinolone-resistant isolates Antimicrobial

No. (%) of susceptible isolates

agent

Escherichia

Klebsiella

Klebsiella

Proteus

Proteus

oxytoca (N

vulgaris

mirabilis

(N = 45)

= 3)

(N = 7)

(N = 2)

coli (N = 126) pneumoniae

Ampicillin

38

30.2

15

33.3

–

–

1

–

–

–

SXT

31

24.6

16

35.6

–

–

3

–

1

–

Gentamicin

60

47.6

19

42.2

2

–

3

–

–

–

Amikacin

92

73.0

23

51.1

3

–

6

–

–

–

Polymyxin B

105

83.3

39

86.7

3

–

–

–

–

–

Tigecycline

36

28.6

24

53.33

2

–

1

–

–

–

Imipenem

93

73.8

40

88.9

2

–

4

–

2

–

Cefotaxime

97

77.0

22

48.9

4

–

5

–

1

–

Ceftazidime

90

71.4

36

80.0

4

–

7

–

1

–

Ceftriaxone

89

70.6

40

88.9

5

–

5

–

2

–

SXT, trimethoprim/sulfamethoxazole.

Table 3 Minimum inhibitory concentrations (MICs) of quinolone-resistant isolates Isolate

MIC range (g/mL) NOR

Escherichia coli

CIP

OFX

WT

Tc WT

Tc

WT

Tc

64

2– 4 to

2

16

0.125– 16

to

4

≥25

pneumoniae

4 to ≥25 6

Tc

WT

0.125– 128

to

6

≥25

≥25

≥25

6

6

6

32

≤0.125 64

4– 1 to 8

WT

MOX

≥25

6 Klebsiella

LVX

2

8 to

0.5

1–2

to

0.5

≥25

≥25

to

to

6

6

≥25

≥25

6

6

≥256 4–8

128

oxytoca

to

to

≥25

≥25

≥25

6

6

6

Proteus vulgaris

Proteus

64 to

4– 128 8

to

2–4

128

32– 256

0.5–1

1–2

1–2

128 to

≥25

≥25

≥25

6

6

6

≥256 4

2

0.125 2

0.125– 128 0.5

≤0.125 1

–

–

≥25 6

≤0.125 1

mirabilis NOR, norfloxacin; CIP, ciprofloxacin, OXF, ofloxacin; LVX, levofloxacin; MOX, moxifloxacin; WT, wild-type; Tc, transconjugant.

–

to

0.125– 4 to 0.15

–

to

Klebsiella

4

Tc

–