Molecular epidemiology of multi-resistant Escherichia coli

Journal of Hospital Infection (1999) 43: 39–48

Molecular epidemiology of multi-resistant Escherichia coli A. Guyot*, S.P. Barrett*, E.J.Threlfall†, M.D. Hampton†,T. Cheasty† *Department of Diagnostic Bacteriology, St Mary’s Hospital, London W2 1NY, †Laboratory of Enteric Pathogens, Central Public Health Laboratory, London NW9 5HT Summary: In this case-control study multi-resistant Escherichia coli isolates were characterized on a molecular level and risk factors for their development were identified. Thirty-two multi-resistant E. coli strains were isolated from the urine of 13 patients attending a renal clinic for chronic urinary tract infection (UTI) and from different sites of 11 terminally ill patients with nosocomial infections hospitalized on five different wards. All 32 isolates were resistant to ciprofloxacin, cotrimoxazole and produced beta-lactamase. All strains contained plasmids of 2–110 MDa of which a 50 MDa and a 100 MDa plasmid were present in 81% of the strains. Pulse-field gel electrophoresis (PFGE) analysis demonstrated 17 genotypes among 32 strains which indicates a polyclonal outbreak with some geographic clustering. Monitoring of patients over the study period showed that either the resident genotype remained the same and that these retained strains underwent changes in their plasmid contents, or that they were replaced by a different genotype after several months of therapy for chronic UTI. Univariate analysis indicated that multi-resistant E. coli develop in the presence of long-term selective ciprofloxacin pressure at a dosing regimen of 250 mg bid for more than 20 days and that treatment with a broad spectrum antimicrobial for more than three days favours the selection of multi-resistant E. coli in the flora of terminally ill patients with multiple disorders. © 1999 The Hospital Infection Society

Keywords: Escherichia coli; ciprofloxacin; antimicrobial drug resistance; molecular epidemiology.

Introduction Fluoroquinolone resistance in Escherichia coli has emerged in Europe and the USA during the 1990s.1,2 Ciprofloxacin resistance rates in E. coli are reported to have risen from less than 1% in Received 10 February 1999; manuscript accepted 16 June 1999 Address correspondence to: Andrea Guyot, Department of Diagnostic Bacteriology, St. Mary’s Hospital, Praed Street, London W2N1Y. E-mail: [email protected]

0195–6701/99/09039 + 10 $12.00

1989 to over 5%, and in a Spanish center even to 18%.3 High level resistance to ciprofloxacin (MIC≥2 mg/L) has been detected in England in 1996 in 6% of E. coli isolates from cases of bacteraemia.4 In St Mary’s Hospital in London, a tertiary referral center, the resistance rate to ciprofloxacin increased from 3·4% in 1996 to 5% in 1998 among coliforms. These ciprofloxacin-resistant coliforms are frequently also resistant to other broad-spectrum antibiotics such as ampicillin (73%), cotrimoxazole (89%), augmentin (37%) cefuroxime (76%) and © 1999 The Hospital Infection Society

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ceftazidime (28%). In St Mary’s Hospital, ceftazidime is usually used to treat infections with multi-resistant coliforms. E. coli is pathogenic particularly in the urinary tract and plays an important role in nosocomial infections. E. coli isolates accounted for 12·7% of intensive care (ICU) acquired infections in 17 western European countries in 1996 and for 13·6% of nosocomial infections in an American hospital surveillance study in 1992.5,6 The present study characterized the molecular epidemiology of multi-resistant E. coli strains and identified risk factors for acquisition.

A. Guyot et al.

ciprofloxacin and ceftazidime by an agar dilution method in DST agar. Serial two-fold dilutions of each antimicrobial drug (Adatabs, MAST Diagnostics, Bootle, Merseyside, UK) were incorporated into the medium ranging for ciprofloxacin from 1–128 mg/L and for ceftazidime from 0·1 to 8 mg/L. Plates were inoculated by a Denley multipoint inoculator with an inoculum of 104 cfu/spot. E. coli NCTC 11560 and NCTC 10418 and a ciprofloxacin-resistant strain with known MIC were used as control strains. Plasmid analysis

Materials and methods Bacterial strains

Thirty-two E. coli strains with resistance to at least ciprofloxacin, ampicillin and sulphamethoxazole were subjected to further characterization in the Diagnostic Bacteriology Department of St Mary’s Hospital, London between December 1996 and June 1998. The strains were identified as E. coli by API 20E (BioMerieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Nineteen isolates were derived from 19 different patients and 13 strains were isolated from consecutive urine samples of five patients who were monitored for chronic urinary track infection (UTI) in the renal transplant clinic. Antimicrobial susceptibility testing

All 32 isolates were screened for resistance to ciprofloxacin, ampicillin, sulphamethoxazole, cefuroxime, gentamicin and tetracycline by disk diffusion on DST agar (MAST Diagnostics, Bootle, Merseyside, UK) using E. coli (NCTC10418) as control strain according to Stokes’ method. Resistance was confirmed by an agar-incorporation breakpoint method, with ampicillin at a concentration of 128 mg/L, sulphamethoxazole at 64 mg/L, cefuroxime at 16 mg/L, gentamicin at 4 mg/L, tetracycline at 123 mg/L and ciprofloxacin at 1 and 4 mg/L. MICs were determined for

Plasmids were extracted by the method of Kado and Liu and separated by electrophoresis in agarose 0·7% w/v gels.7 The molecular sizes of plasmids were estimated by comparison with plasmids of known molecular mass. The plamids were prepared on repeated occasions. Detection of β-lactamase

Inducible cephalosporinase was detected by double disk diffusion by placing a 30 µg imipenem disk (MAST diagnostics) beside a 30 µg ceftazidime (BBL, Becton Dickinson, Oxford, UK) and observing a flattened inhibitory zone around the ceftazidime disk opposite the imipenem disk.8 Isolates were screened for ESBL by double disk diffusion.9 Enhancement of the inhibitory zone between a clavulanate impregnated disk (Augmentin: 20 µg amoxicillin plus 10 µg clavulanic acid) and a ceftazidime disk placed 2 cm apart was interpreted as indicating the presence of an extended-spectrum β-lactamase. PFGE

Total genomic DNA was prepared as described by Powell et al.10 Slices of agarose blocks containing the DNA were digested twice overnight with restriction enzyme Xba1 (Boehringer Mannheim, Germany). A lambda ladder (New England Biolabs Inc.) served as size marker. DNA fragments were separated in 1% w/v agarose gel in 0·5× TBE buffer at 12°C using a

Molecular epidemiology of multi-resistant Escherichia coli

CHEF-DRII apparatus (Bio-Rad, Richmond, Ca). The pulse time was ramped from 5–60 s for 48 h at 5·85 V/cm. Gels were stained with 0·4 µg/mL ethidium bromide and illuminated with UV light and photographed. To eliminate the influence of large plasmids on the PFGE pattern, only fragments larger than 160 kb were scored in the numerical analysis. Faint bands representing partial digest products were not considered in the analysis. All strains were run at least twice. The dendrogram was constructed according to the unweighted pair group method using Arithmetric Averages (UPGMA) after calculating the Dice coefficient for each pair of pulsedfield patterns. Case control study

Case notes of 21 infected patients were available for review. Eleven cases from the renal transplant clinic were age and sex matched with 11 renal clinic patients from whose urine a ciprofloxacin-sensitive E. coli had been isolated during the study period. Ten infected cases from the wards were matched with 10 patients on the same wards in whom a ciprofloxacin sensitive E. coli had been found in similar infection sites during the study period. Statistics

Patients’ characteristics for cases and controls were compared by univariate analysis using the χ2 Fisher’s exact test for categorical variables (Epi Info). A two-tailed P < 0·05 was considered statistically significant. Differences in strains from the renal clinic and wards were tested by Fisher’s exact test.

Results Strains and patients (Table I)

Thirty-two ciprofloxacin-resistant E. coli were isolated from 24 patients of whom five were monitored for resistant urine isolates for periods of 1–8 months. Twenty-four strains (72%)

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were isolated from the urine of 16 patients with chronic complicated UTI, three from blood culture, two from sputum, two from drain fluid and one from skin. Of the 24 urine isolates, 21 were from 13 patients of the renal transplant clinic (53%), which nine patients were attending after having received a renal transplant and the other four for end-stage renal failure (ESRF). The remaining isolates came from five patients admitted on to the surgical ward, two patients on to intensive care unit (ITU), two patients on the renal ward, one patient from the vascular surgical ward and one from the neurology ward. Antimicrobial resistance

All 32 E. coli strains were resistant to ciprofloxacin (MIC≥4 mg/L), ampicillin (MIC > 128 mg/L) and sulphamethoxazole (MIC > 64 mg/L). Seventeen isolates demonstrated resistance to ceftazidime (MIC≥8 mg) and additional three possessed an inducible cephalosporinase with baseline MICs for ceftazidime of 0·25–0·5 mg/L. By the double disk diffusion method, 19 strains produced an ESBL which was inhibited by clavulanic acid. Ceftazidime-resistant strains were isolated from inpatients (10/12) more frequently than from patients from the renal transplant clinic (7/21)(P=0·004, Fisher’s exact test). Twenty isolates were resistant to gentamicin (MIC≥4 mg/L) and 21 were resistant to tetracycline. All strains remained susceptible to carbapenems and to the combination piperacillin plus tazobactam. Serotypes, plasmid contents and PFGE profiles are shown in Figures 1–3. Fifteen of 32 strains were not typable for O-antigen and the remaining 17 were found to belong to ten different serogroups Every strain contained between one and six plasmids with sizes of 4–110 MDa (6·4–176 kb). A 100 MDa (160 kb) and a 50 MDa (80 kb) plasmid were the most common, being present in 18 and 21 isolates, respectively, 12 strains possessing both. Altogether there were 25 plasmid patterns among the 32 strains. The plasmid pattern could differ within a serogroup (strains 7 and

42

A. Guyot et al.

Table I

Characteristics of strains – continued opposite Antimicrobial resistance

1

2

No

Date

Name

Spec

Loc

PFGE serotype

1a 1b 1d 1e 2a 2b 3a 3c 3d 12a 12b 23a 23b 4 29 15 24 28 8 20 31 19 22 25 27 33 30 10 5 6 32 7

16·12·96 16·3·97 30·5·97 7·8·97 1·3·96 13·1·97 16·12·96 7·8·97 25·9·97 18·7·97 13·2·98 31·12·96 6·7·98 7·2·97 13·1·98 3·9·97 5·3·98 15·3·98 29·4·97 11·9·97 30·4·98 2·9·97 5·2·98 7·3·98 12·3·98 8·6·98 23·4·98 8·5·97 24·2·97 4·3·97 20·5·98 1·4·97

Pat 1 Pat 1 Pat 1 Pat 1 Pat 2 Pat 2 Pat 3 Pat 3 Pat 3 Pat 4 Pat 4 Pat 5 Pat 5 Pat 6 Pat 7 Pat 8 Pat 9 Pat 10 Pat 11 Pat 12 Pat 13 Pat 14 Pat 16 Pat 17 Pat 18 Pat 19 Pat 20 Pat 21 Pat 22 Pat 23 Pat 24 Pat 25

U U U U U U U U U U U U U U U U U U U U U DRF SKIN U DRF BLC BLC U SPT DRF BLC U

RC RC RC RC RC RC RC RC RC RC RC RC RC RC RC RC RC RC RC RC RC RW RW DWR DWR DWR DWR DWR LiHo ITU ITU ZCO

A1 A2 A1 A1 B1 B2 A1 A1 A6 B3 B3 A12 A12 A1 A4 E B4 A5 A7 A9 A1 A8 A10 A10 A10 A10 A13 A12 D D A9 E

0 0 0 170 23 0 0 0 0 0 – – 8 0 51 130 1 0 160 101 – 0 91 0 91 0 0 8 0 99 101 130

R

E

S

I

ind. Ceph.5

AM6

plasmid profile

R-pattern

CIP MIC3

CAZ MIC4

pA1 pA2 pA3 pA3 pB1 pA4 pA3 pA3 pB2 pC pC pD1 pD2 pA3 pA8 pA8 pB3 pB3 pA6 pE4 pA7 pA2 pA9 pA10 pA9 pA10 pG pA6 pA7 pA5 pA6 pF1

R1 R2 R1 R1 R11 R4 R2 R3 R12 R6 R3 R2 R2 R2 R5 R5 R9 R9 R1 R7 R11 R6 R9 R9 R9 R9 R13 R8 R7 R7 R9 R9

128 128 128 128 256 256 128 128 64 4 1 256 256 128 32 64 128 128 128 32 256 32 128 128 128 128 16 64 64 64 128 64

16 0·5 16 16 2 16 1 1 16 2 1 1 1 0·25 0·5 0·25 16 32 16 16 1 2 16 16 16 16 0·25 0·5 16 16 16 16

pos pos

pos

r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r

1

spec.=specimen type U=urine DRF=drainfluid SPT=sputum BLC=blood culture loc=location RC=renal clinic RW=renal ward DWR=surgical ward LiHo=neurology ward ITU=intensive care unit ZCO=vascular ward 3 CIP=ciprofloxacin, 4CAZ=ceftazidime, 5 inducible cephalosporinase, 6AM= ampicillin, 7AUG=coamoxyclav, 8 extended spectrum β lactamase, 9SUL=sulfamethoxazole, 10GEN=gentamicin, 11TET=tetracycline 2

15). A relationship between antimicrobial resistance pattern and plasmid profile was not apparent. Overall 26 different strains were characterized differing in PFGE profile, plasmid contents, resistance pattern and serotype. Seventeen different PFGE profiles were found among the 32 strains. The genotypes were in agreement with the serogroups except

for one follow-up case (patient 1) in whom the genotype remained the same but changed from non typable (strain 1d) to serogroup 0170 (strain 1e). Clustering was observed on a surgical ward and in the renal transplant clinic. Four isolates with PFGE profile A10 (Figure 3) and a similar plasmid content were isolated from three patients on the surgical ward and one patient on the renal ward within a period of

Molecular epidemiology of multi-resistant Escherichia coli

43

Antimicrobial resistance S

T 7

AUG

ESBL

s s s s r s s s r s s s s s s s s s s s r s s s s s s s s s r s

pos pos pos pos

pos pos pos

pos pos pos pos

pos pos pos pos

pos pos pos pos

A 8

N 9

SUL r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r

T 10

GEN r r r r s s r s r s s r r r s s r r r s s s r r r r r r s s r i

P 11

TET s s s s r s s s s r s s s s s r r r s r r r r r r r r r r r r

100 MDa

L 70–90

A

S

M

I

S

60–70

50

40

30

<20

100

50 50 50 50 60 50 50 50 60

100 100 72 100 100 r

100 100 100 100 100 100 100 100 r

100 100 100 100 100

four months. A strain with PFGE profile D and serogroup 099 was found in two patients who were admitted to two different wards at the same time. Monitoring of urine isolates from patients of the renal transplant clinic showed that three patients (1, 2 and 3) retained the same strain A1 over 7 months. In two patients (2 and 3) the genotype changed within one month and in two cases (patients 1 and 5) the

60 60

50 50 50

4 4

4

4 4 4

30 30 38 38

11

38 38 80 87 80 90

80 80

50

42

50 50 50 50 50 50

42

50 50 50 50

42 26

33 33

29 26 26 26 26

4 6 4 4 4 4 4 4

26 42

4

66

same strain had acquired (A12) or lost plasmids (A2) after 3–7 months. Case-control study (Table II)

The renal clinic case group contained eight transplant recipients and three patients with ESRF, and the control group six transplant patients and five ESRF cases. The case group and control

44

A. Guyot et al.

Arrow indicates size of largest plasmid.

Figure 1 PFGE profiles

First and last lanes are plasmids of known size of an E.coli control strain. Figure 2 Plasmid profiles

Molecular epidemiology of multi-resistant Escherichia coli

group from the wards contained three sputum, two drain fluid, two urine, two blood culture and one wound isolates, respectively. All renal clinic patients had received ciprofloxacin in the previous 12 months whereas only four inpatients had a history of fluoroquinolone therapy. Eight renal patients of the case group received ciprofloxacin at a dose of 250 mg for more than 20 days in the previous 6 months which appears to be the strongest risk factor for the selection of ciprofloxacin resistant E. coli in renal clinic patients (odds ratio 100, 95% confidence interval:

45

4 < OR < 15102, P=0·0006). Renal transplantation and immunosuppression did not play a significant role. Obstructive urinary tract pathology was a risk factor for carriage of multiresistant coliforms (OR=24, P= 0·01) but not specifically for ciprofloxacin resistance. Risk factors for infection with ciprofloxacinresistant multiple-resistant E. coli in admitted patients were less obvious. The strongest risk factor appeared to be use of a broad-spectrum antibiotic (cefuroxime, ceftazidime, co-amoxyclav or ciprofloxacin) for more than three days

Genotype Serotype Strain No 0 A2 1b A4

051

29

A8

0101

20.32

A5

0

28

A1

0(0170) 1a,1d,1e13a 4,31 0 3d

A6

A7

0.36

0.53

0.69

0.84

Figure 3 Dendrogramm showing relatedness of strains according to PFGE profiles.

0160

8

A13

0

30

A10

091

22,27(25,33)

A8

0

19

A12

06

10,23b23e

E

0130

7,15

B1

023

2a

B2

0

2b

B3

0

12a,12b

B4

01

24

0

99

5,6

1.00

46

A. Guyot et al.

Table II

Case–control study in renal transplant clinic

N Age (x ± s) Sex (F,M) 250 mg cip bid for >20d in previous 6 months Transplant recipient Immuno-suppressive drugs Obstructed urinary tract

Carrier of cip resistant E. coli

Carrier of cip susceptible E. coli

11 50·2 ± 11 9,2 10

11 48 ± 14 9,2 1

8 9 9

6 9 7

Odds ratio with confidence interval

X2 Fisher’s exact test

100 (4
P=0·0006 P=0·65 P=1 P=0·63

Cip=ciprofloxacin

Table III Case-control study for patients on the wards

N Age (x ± s) Sex (F,M) >3 admissions in previous 5 years Hospital stay >30 d before isolation of multires. E. coli Usage of broad spectrum antibiotic >3 d in previous month Death within 1 months after isolation of multires. E. coli MRSA carrier

Carrier of cip resistant E. coli

Carrier of cip susceptible E. coli

Odds ratio with confidence interval

X2 Fisher’s exact test

10 68·2 ± 12·8 3,7 7 4

10 67·7 ± 13·4 3,7 3 1

OR=5·44 OR=6

P=0·179 P=0·3

6

1

P=0·03

7

3

3

3

OR=13·5 (0·92OR>63) 1

in the previous four weeks (OR=13·5 confidence interval 0·9
Discussion This study identifies two patients’ group infected or colonized with ciprofloxacin multiple-resistant coliforms. One comprises of renal clinic outpatients who either received a transplant or are in ESRF, and the other group are

P=0·179 P=1

terminally ill patients with multiple disorders admitted to wards of a teaching hospital. PFGE, plasmid-analysis and serotyping indicate a polyclonal outbreak with 17 different PFGE profiles, 25 plasmid patterns and at least 10 serotypes. Some clustering could be recognized in isolates from the wards (PFGE profiles A10 and D). PFGE type D and A10 occurred on different wards in the same time period. As some of the patients who carried these strains had never been nursed on the same ward together transmission may have occurred by shared equipment or staff. Horizontal spread of Enterobacteriaceae causing UTI was reported on an acute medical ward in a London hospital in 1993.11 In that outbreak transmission could not solely be ascribed to a shared nursing team. In the renal clinic, 14 strains differing in genotype, plasmid profile, antibiotic resistance pattern and serotype were isolated from 12

Molecular epidemiology of multi-resistant Escherichia coli

patients. This indicates that the endogenous resident intestinal flora developed drug resistance rather than cross-infection was the main problem. Strains with same genotype which were collected from the same patient over seven months underwent changes in their plasmid content and strains, which differed in their genotype, had been isolated from two renal clinic patients (2 and 3) within one month. Early serotype studies on intestinal E. coli isolates during treatment of UTIs showed that the development of resistance was not due to the original E. coli serotype becoming resistant but to the resident flora being replaced by a new resistant serotype.12 After treatment, loss of resistance in E. coli was a result of colonization of the bowel with a different drug-sensitive serotype. Our data suggest that the resident resistant E. coli strains can undergo changes in their plasmid contents (1 and 5) or resistance pattern (Patient 4) and that more than one PFGE type can develop resistance (Patients 2 and 3). It is possible that the previously isolated genotype was still resident but not isolated on the second occasion. We found the strongest risk factor for the development of fluoroquinolone resistance among renal clinic patients was a dosing regimen of 250 mg ciprofloxacin bid for more than 20 days. In other studies an AUIC (area under the inhibitory curve) of 100 or Cmax/MIC < 5:1 was associated with the development of ciprofloxacin resistance in Enterobacteriaceae.13 In our study ciprofloxacin resistant E. coli were not isolated from patients who received this dosing regimen for only seven days. High level ciprofloxacin resistance is based on at least two mutations in the chromosomal gyrA gene and the expression of membrane porins, which facilitate the influx or efflux of ciprofloxacin.14,15 It appears if ciprofloxacin is administered at this low dose, the MIC may rise by stepwise mutation until the Cmax/MIC ratio falls below 5:1. In an American hospital-based study, prior receipt of fluoroquinolones was the strongest risk factor for acquisition of ciprofloxacin-resistant coliforms among patients of the long term care facilities.16 As on the acute care wards the majority of patients from whom

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ciprofloxacin-resistant coliforms were isolated had not received a fluoroquinolone and many isolates belonged to a single species, horizontal spread was assumed. Our data show that there is a significant association between the usage of one of the broad-spectrum antimicrobials cefuroxime, co-amoxyclav, ceftazidime and fluoroquinolone for more than three days in the previous month before isolation selects ciprofloxacin and multiple-resistant E. coli in the flora which colonizes admitted patients. An earlier study of our department identified a large conjugative plasmid of 105–120 kb in Enterobacteriaceae isolated from renal unit patients as the cause for transmissible multiple resistance and the use of aztreonam as driving force for the spread of this plasmid among different Gram-negative species.17 In the present investigation only 57% of E. coli contained a 160 kb (100MDa) large plasmid and 66% a 80 kb (50MDa) plasmid. Resistance to aztreonam and 3rd generation cephalosporins in E. coli is usually based on a mutation in the TEM gene. The TEM gene is part of transposon 2 and in Gram-negative bacteria usually inserted on conjugative plasmids but can also be found in chromosomal DNA. Thirty-one isolates harboured plasmids of at least 30 MDa making plasmid mediated β-lactam resistance possible. Plasmid encoded high level ciprofloxacin resistance has not yet been reported. Hence in the present study the development of multiple resistance cannot be solely explained by conjugative spread of a plasmid. Our results suggest that in the face of longterm antibiotic usage E.coli strains of different genotypes can undergo multiple mutations and acquire resistance genes which can be lost when the selective pressure ceases. Cross-transmission and antimicrobial usage favour the colonization and infection with multi resistant coliforms of terminally ill patients on the wards.

Acknowledgement We thank Dr D. Taube, director of the renal clinic at St. Mary’s Hospital, for permission to perform the case-control study.

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