Molecular epidemiology and antibiotic resistance of Enterobacter spp. from three distinct populations in Grampian, UK

Molecular epidemiology and antibiotic resistance of Enterobacter spp. from three distinct populations in Grampian, UK

International Journal of Antimicrobial Agents 20 (2002) 419 /425 www.isochem.org Molecular epidemiology and antibiotic resistance of Enterobacter sp...

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International Journal of Antimicrobial Agents 20 (2002) 419 /425 www.isochem.org

Molecular epidemiology and antibiotic resistance of Enterobacter spp. from three distinct populations in Grampian, UK F.M.E. Wagenlehner a,, F.M. MacKenzie a, K.J. Forbes b, I.M. Gould a a

Department of Medical Microbiology, Aberdeen Royal Infirmary, Aberdeen, UK b Department of Medical Microbiology, Aberdeen University, Aberdeen, UK Received 1 February 2002; accepted 3 April 2002

Abstract The distribution of Enterobacter spp. within the population of Aberdeen Royal Infirmary was compared with the outpatient population with regard to molecular epidemiology and antibiotic resistance. Enterobacter spp. from 60 patients and one environmental site were characterised as ITU, non ITU and outpatients’ isolates. Thirty-five percent were blood culture isolates. Cefotaxime resistant strains in the hospital were frequent. Cefotaxime (64%) sensitive isolates were inducible for hyperproduction of Bush group 1 b-lactamase. Isolates were further investigated by PFGE. Isolates (27%) were clonally related and typed in four clusters. Consecutive isolates were studied in selected patients showing minor genomic changes. One environmental isolate from a deep sink at ITU was related to a patient’s isolate. # 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Enterobacter spp.; Clonal relationship; Antibiotic resistance

1. Introduction Enterobacter spp. have been recognised as increasingly important nosocomial pathogens in recent years [1,2]. In particular, patients with a severe underlying illness, who are on the ITU and who are immunosuppressed (i.e. after organ transplantation) are more prone to acquire infection. Enterobacter spp. can cause lower respiratory tract infections, abdominal infections, urinary-tract infections, soft tissue infections, endocarditis, bacteraemia, central nervous system infections, ophthalmic infections as well as infections of bone and joints [2]. More recently, it appears that Enterobacter spp. have spilled over into the community colonising the intestine of otherwise healthy individuals. The majority of infections arise endogenously from the flora of patients who have become chronically colonised [3]. In  Corresponding author. Present address: Urologische Klinik, Klinikum St. Elisabeth, St. Elisabeth-Str. 23, 94315 Straubing, Germany. Tel.: /49-9421-710-1700; fax: /49-9421-710-1717 E-mail address: [email protected] (F.M.E. Wagenlehner).

addition, however, cross-infection has been reported on neonatal intensive care units, burn units, transplantation units and surgical wards [1,4 /6]. A high percentage of these organisms are innately resistant to older antimicrobial agents and have the ability to rapidly develop resistance to newer agents. b-Lactam antibiotics such as the third-generations cephalosporins are particularly good at selecting for highly resistant strains [7 /9]. Enterobacter spp. have been found to account for 4/ 12% of sepsis caused by Gram-negative organisms [10 / 12], accompanied by a mortality rate ranging from 15 to 87% [2]. The disparate rates result from differences in the populations of patients studied and a distinct prevalence of resistance amongst them and different geographical areas. The aim of this study was to compare Enterobacter isolates from patients at Aberdeen Royal Infirmary (ARI) in the north /east of Scotland and the local community by looking at their antibiotic resistance patterns, b-lactamase production and PFGE genotypes. The results will contribute to the further investigation of the epidemiology of this species.

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2. Materials and methods 2.1. Bacterial strains Enterobacter spp. strains were collected from four distinct sources: (i) ARI intensive therapy unit (ITU), including the coronary ITU, (ii) non ITU isolates from various hospital sources, (iii) isolates from outpatients and (iv) one isolate from a deep sink of a washing basin from ITU. The hospital strains were collected over a period of 19 months (January 1997 /July 1998), community isolates over 3 months (May 1998/July 1998) and environmental isolates from ITU once in July 1998. Enterobacter cloacae ATCC 23355 was used as a comparator. Isolates were maintained at /70 8C in Microbank (PRO-LAB Diagnostics, Canada) storage tubes. 2.2. Biotyping Presumptive Enterobacter spp. isolates from community samples were screened with a set of indole, methylred and Voges /Proskauer reactions. Full biochemical identification was determined by the API 20E system (Api-bioMe`rieux) according to the manufacturer’s instructions [20]. 2.3. Susceptibility testing Minimum inhibitory concentrations (MICs) of cefotaxime, gentamicin and ciprofloxacin were determined by the NCCLS agar dilution method [13] with Mueller/ Hinton agar and an inoculum of 104 cfu per spot. Escherichia coli ATCC 25922 was used as the control organism. 2.4. Bush group 1 b-lactamase hyper production For assessment of inducible hyper production of the chromosomally encoded Bush group 1 b-lactamase [14], imipenem (10 mg) and clavulanic acid (10 mg), known to be strong inducers of the enzyme, were used. Discs containing amoxycillin/clavulanic acid: 2/1 (20 mg/10 mg), cefotaxime (30 mg) and imipenem (10 mg) were placed 20 mm apart with cefotaxime in the middle. Flattening of the concentric circular zones around cefotaxime was regarded as antagonism [15]. 2.5. Chromosomal analysis by PFGE Unsheared DNA was prepared as described by Smith et al. [16] with minor modification. In brief, cells were grown on blood agar, harvested by disposable loops, twice suspended in 1000 ml of suspension buffer (100 mM Na2 /EDTA, 10 mM EGTA, 10 mM Tris /HCl, pH 8.0) to a density of 3/108 cells per ml, and collected by

centrifugation. The cell pellet was thoroughly resuspended in 700 ml of the same buffer. Suspension (75 ml) was mixed with 200 ml of preheated 1% low-meltingpoint InCert agarose (FMC BioProducts, Risingevej, Denmark) and pipetted into 200 ml insert moulds. The inserts were incubated at 4 8C for 15 min. and then extruded into 1000 ml of lysozyme solution (2 mg of lysozyme per ml in 10 mM Tris /HCl /1 M NaCl /100 mM Na2 /EDTA /0.5% cetyl ether /0.2% Nadeoxycholate/0.5% N -lauryl-sarkosine, pH 7.5). The samples were incubated for 4 h at 37 8C with gentle shaking (80 rpm) followed by a short rinse in washing solution (10 mM Tris /HCl, 1 mM Na2 /EDTA, pH 8.0). This was replaced by 1000 ml of Proteinase K solution (1 mg of Proteinase K per ml in 0.5 M Na2 / EDTA /25% Na2 /dodecylsulfate, pH 8.0) and the inserts incubated at 50 8C for 14 h with gentle shaking (80 rpm). The inserts were washed eight times in washing solution (10 mM Tris /HCl, 1 mM Na2 / EDTA, pH 8.0) and stored at 4 8C in the same solution. For restriction endonuclease digestion, thin slices were cut off the agarose plugs and each slice equilibrated at 37 8C for 4 h in 200 ml incubation buffer (1 ml bovine serum albumin 1 mg/ml /20 ml 10 / restriction-buffer (Pharmacia One-for-All) /5 ml 20 mM Dithiothreitol /3 ml digestion enzyme /171 ml distilled H2O). All samples were digested with Xba I and isolates which were similar or related were additionally digested with Spe I . Stop buffer (0.25% bromophenolblue /0.1% Na2 /dodecylsulfate) (50 ml) were added to each insert to terminate digestion. The solid plug-samples were then loaded into the wells of a 1.0% (wt/vol) agarose gel (Fast-lane, FMC, Risingevej, Denmark) prepared in running buffer (0.5 /TBE). A similarly treated plug of E. cloacae ATCC 23355 strain and lambda ladder (New England Biolabs) were used as standards. PFGE was performed in a CHEF-Mapper electrophoresis chamber (Bio-Rad., Munich, Germany) at 14 8C at 200 V. For digestion with Xba I pulse time was linearly ramped from 5 to 50 s over 20 h, and for Spe I digestion, pulse time was linearly ramped from 1 to 30 s over 18 h. Electrophoretic images were scanned and further evaluated by computer soft ware using the criteria of Tenover et al. [17]. 2.6. Environmental investigation The sinks and drains in ITU were swabbed with sterile swabs and cultured on MacConkey agar. Where Gramnegative bacteria growth was evident screening and identification methods, as mentioned above, were used to identify Enterobacter spp. 2.7. Statistics For statistical analysis the x2-test was employed.

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3. Results 3.1. Body sites and organisms Sixty primary isolates from 60 patients were studied (Tables 1 and 2) and categorised into ITU, non-ITU and community isolates, the clinical source and whether a blood culture isolate, species identification determined by API. Twelve of 60 (20%) of isolates could be identified only to the genus level by API system. Twenty of 60 (35%) of isolates were from blood cultures or central lines. There were no community isolates from blood cultures. 3.2. Antibiotic resistance The MIC results for gentamicin, cefotaxime and ciprofloxacin are shown in Table 2. NCCLS breakpoints for susceptibility to gentamicin, cefotaxime and ciprofloxacin ( 5/4, 5/8 and 5/1 mg/l, respectively) were employed. Thirty-eight of 60 (63%) isolates were resistant to cefotaxime. Outpatient isolates were significantly less resistant than nosocomially acquired isolates (P B/ 0.05) (3/9 vs. 35/51). Fourteen of 22 (64%) cefotaxime susceptible isolates phenotypically demonstrated induction of a hyper produced, chromosomally encoded Bush group 1 b-lactamase, 8/22 (36%) isolates were noninducible. Three of 60 (5%) isolates were resistant to ciprofloxacin and notably, all three isolates had been nosocomially acquired. All isolates were susceptible to gentamicin. 3.3. PFGE In 12 patients duplicate or triplicate consecutive sampled isolates were evaluated, but are not shown in

Fig. 1. PFGE patterns after digestion with Spe I of four Enterobacter spp. isolates. Three consecutive isolates Enterobacter spp. of a patient’s hip fluid (lanes 1 /3) and one isolate Enterobacter spp. from a distinct patient’s tracheal aspirate (lane 4) belonging to cluster 2. PFGE patterns show one band difference in each isolate.

Fig. 2. In seven patients isolates were taken from the same sites, in five patients isolates were from different sites. These successive isolates showed only minor differences in PFGE patterns with Xba I digestion whereas digestion with Spe I was sometimes more precise. Three consecutive Enterobacter spp. from a patient’s hip and one Enterobacter spp. from a distinct patient’s tracheal aspirate revealed one to two bands differences in their PFGE pattern after digestion with Xba I, the last of the triplicate isolates being indis-

Table 1 Clinical source and API identification of test isolates Group

Sample source

ITU

Blooda Respiratory tractb Abdomenc Urine Blooda Respiratory tractb Abdomenc Urine Wounds Joints Urine Faecesd /

Non ITU

Outpatients Total a b c d

Enterobacter spp.

6 1 2 1

1 1

12

Blood cultures and central lines. Lower respiratory tract 14 ; upper respiratory tract 1 . Peritoneal and retroperitoneal cavity. Not isolated as pathogen.

E. cloacae

E. aerogenes

4 4 3

1

12 3 2 2 2 1 8

2

41

E. agglomerans

1 2

1 6

1

Total 5 10 3 1 16 5 4 2 3 2 8 1 60

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Table 2 Summary of PFGE patterns and dendrogram of 60 patients’ primary isolates after digestion with Xba I Number

Group

Isolate reference

Species

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Non ITU ITU Outpatient Non ITU Outpatient Outpatient Non ITU ITU ITU ITU Non ITU Non ITU Non ITU Non ITU Non ITU Non ITU ITU Outpatient Non ITU Non ITU Outpatient Non ITU Non ITU Non ITU ITU ITU Non ITU ITU Outpatient Non ITU Non ITU Non ITU Non ITU Non ITU ITU Non ITU Non ITU Non ITU Outpatient Non ITU ITU ITU Non ITU Non ITU Non ITU Non ITU Outpatient Outpatient Non ITU ITU Non ITU ITU ITU ITU Non ITU Non ITU ITU ITU ITU ITU

CTX 1526 ITU 1489 COM 841/26 BLO 14 COM 383/11 COM 213/11 BLO 30 ITU 1468 ITU 1436 ITU 1483 CTX 1663 BLO 24 BLO 6 BLO 20 COM 281/3 CTX 1525 ITU 1382 COM 852/6 CTX 1533 CTX 1620 COM 699/26 BLO 15 BLO 25 BLO 27 BLO 32 ITU 1527 CTX 1603 ITU 1373 COM 390/7 CTX 1548 CTX 1617 CTX 1658 BLO 1 BLO 7 ITU 1405 CTX 1612 CTX 1626 BLO 2 COM 968/10 COM 752644 ITU 1622 ITU 1623 CTX 956/21 CTX 1541 CTX 1512 BLO 4 COM 645/20 COM 113/18 BLO 29 BLO 33 CTX 1677 BLO 34 ITU 1512 BLO 36 BLO 26 BLO 23 ITU 1383 ITU 1419 ITU 1446 ITU 1417

E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. aerogenes E. cloacae Enterobacter E. cloacae E. cloacae E. aerogenes E. aerogenes E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae Enterobacter spp. E. cloacae E. cloacae Enterobacter spp. E. cloacae Enterobacter spp. E. aerogenes Enterobacter spp. E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae Enterobacter spp. Enterobacter spp. E. cloacae E. cloacae E. agglomerans Enterobacter spp. Enterobacter spp. E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. aerogenes E. cloacae E. aerogenes E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae E. cloacae Enterobacter spp. Enterobacter spp. Enterobacter spp.

Blood

 

    

   

 



      

MIC GENT

MIC CTX

MIC CIP

0.25 1 0.5 0.25 0.5 0.125 0.25 0.25 0.25 1 0.5 0.25 0.25 0.5 0.5 0.25 0.25 0.25 0.25 0.25 0.5 0.5 0.5 0.25 0.25 0.5 0.25 0.25 0.5 0.25 0.25 0.5 0.25 0.25 0.5 0.25 0.5 0.25 0.5 0.5 0.5 0.25 0.5 0.5 0.25 0.25 0.5 0.5 0.25 0.25 0.25 0.25 0.5 0.25 0.25 0.25 0.5 0.5 1 1

16 0.5 64 8 0.25 4 1 16 0.5 1  64 32  64 0.5  64 8 1 0.125 64 32 64  64 2 0.5 32 64 64 16 0.25 64 64  64 32 16 0.25 64  64 0.5 0.25 0.0315  64  64 32  64 32 64 32 0.5 2  64 16  64 8  64  64  64 4 64 16 64

0.06 0.031 0.01 0.01 0.002 0.06 0.01 0.031 0.01 0.031 4 0.1 0.5 0.01 0.06 0.06 0.5 0.008 0.25 0.1 0.031 0.01 0.25 0.5 0.01 0.06 0.031 0.1 0.008 0.01 0.25 1 0.25 0.01 0.01 2 0.5 0.1 0.01 0.01 0.1 0.1 0.25 2 0.25 0.25 0.1 0.008 0.25 0.25 0.1 0.25 0.5 0.25 0.25 0.25 0.1 0.1 1 0.1

Clusters

4 4 4

3 3 2 2

1 1 1 1 1 1 1 1 1

Division into clusters was by genetic identity deduced from dendrogram and evaluation of PFGE patterns using the criteria of Tenover et al. [17]. Positive blood cultures, minimal inhibitory concentration (MIC, mg/l) to gentamicin (GENT), cefotaxime (CTX) and ciprofloxacin (CIP) are denoted.

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Fig. 2. PFGE of Xba I digests of genomic DNA of Enterobacter spp. Dendrogram constructed in BioNumerics† (Applied Maths, Belgium) using UPGMA and Dice% coefficients of similarity. Isolate code numbers are indicated. Cluster 1 (Table 2) indicated with dots.

tinguishable to the distinct patient’s isolate. Digestion with Spe I in this case was more discriminatory, showing one band difference in these isolates (Fig. 1). It may be

that these slight differences were due to differences in plasmid profiles rather than to chromosomal differences. In either event these were often only single or few

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band differences, and by the criteria of Tenover et al. [17] these would be regarded as the same strain. The PFGE patterns and dendrogram constructed for primary patients’ isolates are shown in Fig. 2. There were clearly many clusters related isolates, often differring by one or two bands. Most notably, however, was a large cluster of containing nine isolates (6 / E. cloacae ; 3 / Enterobacter spp.), of which seven were from ITU and two were from elsewhere in the hospital. There were four blood isolates, three lower respiratory tract isolates, one peritoneal isolate and one urine isolate. All were susceptible to ciprofloxacin, seven out of nine were resistant to cefotaxime and one of the cefotaxime susceptible isolates demonstrated inducible, hyper production of a Bush group 1 b-lactamase. Indeed in general clustered isolates were significantly (P B/0.025) more resistant to cefotaxime than those isolates with individual typing patterns (14/16 vs. 24/44).

4. Discussion Enterobacter spp. is known for its propensity to develop antimicrobial resistance during therapy, especially to the third-generation cephalosporins. The reason for this is the ability of these organisms to constitutively produce a chromosomally encoded Bush group 1 blactamase [14] which has good activity against penicillin, piperacillin, ceftazidime, ceftriaxone and cefotaxime and cannot be inhibited by clavulanic acid. This enzyme is typically produced in small amounts, but has a low maximum hydrolysis rate. Thus resistance to the abovementioned antibiotics is conferred only when it is produced in large quantities [7]. Constitutive, hyper production of this enzyme results, from a mutation in the amp D gene, which occurs spontaneously at rates as high as 104 /106 [18]. Sixty-four percent of all our cefotaxime sensitive isolates were inducible for hyper production of this enzyme, 36% were non-inducible. In patients with high-density infections such as deep-seated abscesses and pneumonia, resistance rates to cefotaxime are highest and are reflected in the nosocomial isolates. The gentamicin MIC was 5/1 mg/l for all isolates, thus resistance rates were very low compared with reports from other countries, where resistance to aminoglycosides exceeded 36% [19]. PFGE showed a good correlation of Xba I with Spe I results, indicating that this is a robust typing scheme [6]. Where sequential isolates were available, it was demonstrated that successive isolates showed only minor differences in PFGE patterns with Xba I digestion whereas digestion with Spe I was sometimes more precise. Three consecutive Enterobacter spp. from a patient’s hip and one Enterobacter spp. from a distinct patient’s tracheal aspirate revealed one to two bands differences in their PFGE pattern after digestion with

Xba I, the last of the triplicate isolates being indistinguishable to the distinct patient’s isolate. Digestion with Spe I in this case was more discriminatory, showing one band difference in these isolates (Fig. 1). One isolate of E. cloacae from an ITU patient’s tracheal aspirate and an environmental sample E. cloacae from a deep sink from ITU revealed similar PFGE patterns, but no similarity with any other patient isolate. The patient suffered from a severe S. epidermidis endocarditis of an artificial heart valve and developed a severe pneumonia with Enterobacter spp. in ITU with fatal consequences. This demonstrates that Enterobacter spp. can survive in a nosocomial environment for some time, representing a possible source for cross transmission. Thirty five percent of isolates stemmed from blood cultures or central lines. This indicates that Enterobacter spp. is a highly pathogenic organism that should be followed up closely and treated efficiently. This study investigated the molecular epidemiology and the antibiotic resistance of Enterobacter spp. in three distinct populations. Infection with Enterobacter spp. is generally a nosocomial phenomenon, although occasionally it is isolated from community samples. Hospitalised patients, especially the severely ill, can become infected by multiple strains and have a rather high risk to acquire blood stream infections. It has been shown, that therapy with broad-spectrum cephalosporins was an independent risk factor to develop resistance to third-generation cephalosporins, whereas quinolone therapy was protective [9]. To reduce further increase of antibiotic resistance in Enterobacter spp. a combined antibiotic therapy with aminoglycosides or quinolones and appropriate hygienic measures should be implemented in the hospital.

Acknowledgements This work was supported by a grant from British Society for Antimicrobial Chemotherapy, which was awarded to F.M.E. Wagenlehner, I.M. Gould, F.M. MacKenzie and K.J. Forbes. We thank Kate Milne and Marrianne Harkins for their contribution and help.

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