C Wendt

S Schütt

A H Dalpke

M Konrad

M Mieth

B Trierweiler-Hauke

M A Weigand

S Zimmermann

K Biehler

D Jonas

First outbreak of carbapenemase

Keywords:

published or not. The documents may come

Introduction

Carbapenemases of the Klebsiella pneumoniae carbapenemases (KPC) type belong to the molecular class A βlactamases [START_REF] Queenan | Carbapenemases: the versatile βlactamases[END_REF]. The first clinical isolate of this enzyme that can hydrolyse carbapenems in addition to all cephalosporins was detected in 1996 in North Carolina [START_REF] Yigit | Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae[END_REF]. Since then, KPC-producing K. pneumoniae has spread with alarming speed, especially in New York and the NY area [START_REF] Bratu | Emergence of KPCpossessing Klebsiella pneumoniae in Brooklyn, New York: epidemiology and recommendations for detection[END_REF][START_REF] Lomaestro | The spread of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae to upstate New York[END_REF][START_REF] Bradford | Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 beta-lactamases in New York City[END_REF][START_REF] Woodford | Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center[END_REF]. After the expansion of KPC-K. pneumoniae along the east coast of the United States, the detection of KPC was reported from other countries beginning in 2005 [START_REF] Naas | Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC in a Klebsiella pneumoniae isolate from France[END_REF]. Isolates were reported from South America [START_REF] Villegas | Colombian Nosocomial Resistance Study Group (2006) First detection of the plasmid-mediated class A carbapenemase KPC-2 in clinical isolates of Klebsiella pneumoniae from South America[END_REF][START_REF] Pasteran | Klebsiella pneumoniae carbapenemase-2, Buenos Aires, Argentina[END_REF], China [START_REF] Wei | Plasmidmediated KPC-2 in a Klebsiella pneumoniae isolate from China[END_REF] and Israel

Escherichia coli [START_REF] Cai | Emergence of Serratia marcescens, Klebsiella pneumoniae, and Escherichia coli isolates possessing the plasmid-mediated carbapenem-hydrolyzing β-lactamase KPC-2 in intensive care units of a Chinese hospital[END_REF], Enterobacter spp. [START_REF] Marchaim | Isolation of imipenem-resistant Enterobacter species: emergence of KPC-2 carbapenemase, molecular characterization, epidemiology, and outcomes[END_REF], Salmonella spp. [START_REF] Miriagou | Imipenem resistance in a Salmonella clinical strain due to plasmid-mediated class A carbapenemase KPC-2[END_REF] and Pseudomonas aeruginosa [START_REF] Villegas | Colombian Nosocomial Resistance Study Group (2007) First identification of Pseudomonas aeruginosa isolates producing a KPC-type carbapenem-hydrolyzing beta-lactamase[END_REF]. Recently, the transposon Tn4401 has been proposed to be at the origin of KPC mobilisation [START_REF] Naas | Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene[END_REF]. Sequence analyses of isolates from America and Europe revealed, besides a large consensus, few variations in the left and right target site duplication and, in two out of five investigated strains, a 100-bp deletion upstream of the KPC-2 coding sequence.

We report the first outbreak of KPC-producing K. pneumoniae in Germany, which affected eight patients, of whom three died from infection. This report describes the outbreak, the infection control measures implemented and the results of molecular investigations to elucidate the origin of KPC in our patients.

Methods

Setting

The University Hospitals of Heidelberg are a 1,600 bed tertiary care centre serving about 52,000 patients per year. All affected patients were nursed in the department of general, visceral and transplantation surgery. The 14-bed interdisciplinary surgical intensive care unit (ICU) is operated in cooperation by the department of anaesthesiology and the department of general, visceral and transplantation surgery. A 14-bed intermediate care unit treats intensive care patients who no longer need mechanical ventilation and patients after transplantation of visceral organs, i.e. liver or renal transplant patients. Patients of these units are routinely screened for methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant enterococci (VRE), but screening for Gram-negative microorganisms is not performed. The infection control team was alerted after three patients were detected who had K. pneumoniae with an unusual resistance pattern isolated from tracheal secretion.

Microbiological methods

Enterobacteriaceae isolated from clinical specimens are routinely tested for antibiotic susceptibility by the Vitek 2 system (bioMérieux, Nürtingen, Germany). Susceptibilities were interpreted using the European Committee for Antimicrobial Susceptibility Testing (EUCAST)/BSAC (v.7) breakpoints. The modified Hodge test was performed as described by Lee et al. [START_REF] Lee | Modified Hodge and EDTA-disk synergy tests to screen metalloβ-lactamase-producing strains of Pseudomonas and Acinetobacter species[END_REF].

Screening specimens for KPC-producing K. pneumoniae were cultured on Columbia blood agar, McConkey agar and chromID extended spectrum β-lactamases (ESBLs, bio-Mérieux). Representative colonies of all suspect enterobacteriaceae were tested by the Vitek 2 system. If the minimum inhibitory concentration (MIC) to ertapenem was elevated, further testing by polymerase chain reaction (PCR) and the modified Hodge test was performed.

Characterisation of bla KPC and Tn4401-like elements

The bla KPC gene was detected using PCR primers encompassing the entire coding region, resulting in an amplicon of 893 bp [START_REF] Bradford | Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 beta-lactamases in New York City[END_REF]. Template DNA for cycling PCR was obtained by using standard DNA isolation procedures (High Pure PCR Template Preparation Kit, Roche). Both strands of the PCR products were then sequenced (BigDye Terminator v1.1 Cycle Sequencing Kit, Applied Biosystems) using the amplification primers and detected on an ABI 310 (Applied Biosystems). The sequencing files were assembled from the resultant chromatograms with the Staden suite (version 1.7.0) software package (Staden, R. 1996. Mol. Biotechnol. 5:233) The sequence data were analysed for similar GenBank entries by the BLAST algorithm [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF].

Precise determination of both Tn4401 insertion sites and the variable deletion between the istB and the downstream bla KPC-2 gene, occurring in Tn4401 elements of different geographical origin, was performed by using the primers as described [START_REF] Naas | Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene[END_REF].

Plasmids were obtained by a standard alkaline lysis method and analysed on a 1% agarose gel. E. coli strain J53 containing the plasmid pMG252 (kindly provided by Dr. G. Jacoby, Lahey Clinic, Burlington, MA 01805) served as the size standard.

Genotyping of isolates

Molecular typing was performed after the digestion of chromosomal DNA with the restriction enzyme speI by pulsed-field gel electrophoresis (PFGE) using the contourclamped homogenous electric field (CHEF) electrophoresis technique and amplified fragment length polymorphism (AFLP) [START_REF] Pfaller | PFGE of chromosomal DNA[END_REF]. Clonal diversity was defined as proposed by Tenover et al. [START_REF] Tenover | Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing[END_REF] and evaluated by Jonas et al. [START_REF] Jonas | Discrimination of Klebsiella pneumoniae and Klebsiella oxytoca phylogenetic groups and other Klebsiella species by use of amplified fragment length polymorphism[END_REF].

Conjugation experiments

The PCR-detectable bla KPC gene locus of K. pneumoniae could be mobilised together with the antimicrobial resistant phenotype into an E. coli J53Az R recipient by transconjugation and subsequent selection on 100 µg/ml Naazide in combination with 100 µg/ml cefotaxime after overnight filter-mating, as described previously [START_REF] Jonas | Plasmid-mediated quinolone resistance in isolates obtained in German intensive care units[END_REF]. Antimicrobial susceptibility testing of the donor, recipient and transconjugants was performed according to the National Committee for Clinical Laboratory Standards (NCCLS) [START_REF]Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobially-Fourth Edition[END_REF].

Isoelectric focussing

The β-lactamases produced by the strains were isolated by ultrasonic treatment [START_REF] Ejlertsen | The β-lactamases of Moraxella (Branhamella) catarrhalis isolated from Danish children[END_REF] and characterised with regard to the isoelectric point by isoelectric focussing on a polyacrylamide gel (ampholytes [pH 3 -10]) and inhibition by clavulanic acid [START_REF] Watanabe | Transferable imipenem resistance in Pseudomonas aeruginosa[END_REF].

Outbreak investigation

All patients who presented KPC-producing K. pneumoniae isolated from any body site were defined as cases. Patients who had been nursed in the same room as a case patient were defined as contact patients. If a contact patient was nursed with a case patient on more than one unit, each contact was counted as a single incident. The location and duration of contacts between patients was identified from the hospitals' electronic information system.

The intervention consisted of isolating the affected patients in single rooms and strict barrier nursing was implemented. Health care workers were informed about KPC by information sheets and short informative meetings.

Prevalence screenings were performed on two different occasions. The first screening included all patients who were nursed in the affected units and the second included all patients who were nursed in the affected department during the preceding 10 days.

If case patients were transferred to other institutions, the health care providers of these institutions were informed about the colonisation status of the patients. Furthermore, laboratory and infection control staff of German health care institutions were informed about the identification of KPC in Germany [START_REF] Wendt | Klebsiella-pneumoniae-carbapenemase in Deutschland nachgewiesen[END_REF].

Results

In January 2008, a K. pneumoniae was isolated from a wound specimen of a patient nursed on the interdisciplinary ICU, which was resistant to all β-lactams, fluoro-quinolones, cotrimoxazole, aminoglycosides and ertapenem, but had susceptible MICs to imipenem and meropenem, as measured by the Vitek 2 system. Due to low the MICs to imipenem and meropenem, a carbapenemase was not suspected at this time. A KPC-2-encoding gene locus was identified after two more patients from the same unit had K. pneumoniae with identical susceptibility patterns isolated from respiratory specimens. Strict isolation measures were implemented and health care workers were informed about KPC-mediated resistance by leaflets and informative meetings.

Despite the introduction of these measures, during the following months, five additional patients were identified, who were colonised or infected with KPC-producing K. pneumoniae. Therefore, we performed prevalence screenings of potential contact patients on two different occasions including 31 and 51 patients, respectively. However, all screened patients were tested negative. The last patient associated with the outbreak left the hospital in June and no additional cases were detected during the following six months. All case patients were kept in strict isolation until they left the hospital.

The index patient had no history of travel or contacts to persons from endemic areas. Therefore, it was assumed that the KPC-positive strain may have been introduced by another patient who remained undetected. Case patients had been treated in the hospital for up to 262 days before colonisation or infection with KPC-producing K. pneumoniae became apparent. During this time, the case patients were transferred several times between different units (2 to 13 times, median 8.5 times). They had 624 contacts to 481 patients before they were treated in isolation.

Noteworthy, among the 17 patients who had more than three contacts to case patients, seven became case patients later. The duration of contacts was between 1 and 960 h, whereby patients who became case patients had longer contact times with other case patients (mean 109.3 h, range 4-480 h) than other patients (mean 52.3 h, range 1-960 h). This difference was statistically significant (p = 0.025).

Almost all of the patients who had been in contact with case patients left the hospital with unknown colonisation status. Thus, unrecognised transfer of the KPC-producing K. pneumoniae to other institutions is possible.

Five months after the last case patient had left the hospital, another KPC-producing K. pneumoniae was isolated, this time from an abscess in a carcinoma patient who had been treated in Greece previously (case 9). Typing revealed that this patient was infected with an isolate that was indistinguishable from the outbreak isolates (Fig. 1). Retrospectively, this patient was treated for an abdominal abscess in November 2007-shortly before the outbreakon the same unit as the index case. Unfortunately, no microbiological samples were taken during this in-patient stay.

All case patients suffered from severe underlying diseases: two of them had solid organ transplantation, four suffered from malignancies, two had complication after surgery and one patient had multiple trauma (Table 1). KPC-producing K. pneumoniae caused infections in three patients (pneumonia, wound infection and sepsis). All of these patients were treated with combinations of antibiotics that included colistin. Unfortunately, none of these patients could be successfully treated. An additional case patient died from her underlying disease.

PFGE and AFLP typing concordantly revealed that all patients were colonised by the same K. pneumoniae strain, with one patient harbouring a subtype of the outbreak clone and another patient (patient 5) harbouring the outbreak clone and, additionally, a subtype that displayed a different appearance on the agar plates (Fig. 1). MICs of the K. pneumoniae isolates to several antibiotics and an additional E. coli isolate from the index patient that was also shown to harbour the bla KPC gene are shown in Table 2.

Sequencing of the bla KPC PCR product allowed to identify a KPC-2 carbapenemase. The isoelectric point of the enzyme was 6.8.

Plasmid preparation from both species revealed cccDNA larger than the plasmid pMG 252 (Fig. 2), which served as an approximate standard with an estimated size of 180 kb [START_REF] Wang | Emerging plasmid-mediated quinolone resistance associated with the qnr gene in Klebsiella pneumoniae clinical isolates in the United States[END_REF]. By use of a non-radioactively labelled bla KPC PCR fragment, the plasmids showed the presence of bla KPC DNA sequences in Southern hybridisation experiments (data not shown). Southern blotting of plasmid preparations with a biotin-labelled bla KPC-2 amplicon as a probe revealed a size larger than the plasmid pMG 252 (data not shown).

A bla KPC -positive plasmid could be successfully mobilised from a K. pneumoniae and the E. coli isolate into an E. coli J53 recipient with a conjugation frequency of 2-3 × 10 -8 . Accordingly, the transconjugant showed a three to larger than 11-fold increase of the MIC as compared to the susceptibility of the recipient in all beta-lactams, cephalosporins and carbapenems tested. No such changes occurred when testing ciprofloxacin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole (Table 3). Of note, in the presence of clavulanic acid, the MIC of all antimicrobials tested decreased by up to more than six dilution steps.

Sequence analysis of the three variable sites of Tn4401 elements described in the literature revealed data identical to plasmid pNYC (GenBank accession number EU176011) for both the clinical E. coli and K. pneumoniae isolate, as well as both resulting transconjugants. The left and right target site duplication with the particular sequence ATTGA was demonstrated by sequencing positions 3969-4398 and 13279-13979 (numbering according to GenBank accession number EU176011). Furthermore, the determination of transposon sequences at positions 10827-11263 revealed the 100-bp deletion upstream of bla KPC-2 .

Discussion

To the best of our knowledge, this is the first outbreak of a KPC-producing K. pneumoniae in Middle Europe. Although cases from other Middle European countries have been reported, all of these were single cases and transmission was not observed [START_REF] Naas | Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC in a Klebsiella pneumoniae isolate from France[END_REF][START_REF] Woodford | Arrival of Klebsiella pneumoniae producing KPC carbapenemase in the United Kingdom[END_REF]. The characteristics of our outbreak-long hospitalisation periods of the respective cases, predominantly respiratory colonisation, high proportion of infected patients and a high overall mortality of infected patients-were similar to other outbreaks described so far.

In an outbreak described by Woodford et al., 24 ICU patients were colonised or infected with KPC-producing K. pneumoniae [START_REF] Woodford | Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center[END_REF]. All infections were nosocomially acquired, with the patients having been hospitalised from 9 to 374 days prior to isolation of the organism. The organisms were isolated predominantly from respiratory secretions, but also from urine and blood. A high proportion, i.e. 14 of the 24 patients, was infected and eight of the infected patients died.

≥320 ≥320 ≥320 ≥320 ≥320 ≥320 ≥320 ≥320 ≥320 ≥320 ≥320
Colistin (E-test) 0.75 0.5

Bratu et al. described outbreaks in two New York hospitals involving 58 patients [START_REF] Bratu | Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium[END_REF]. Forty-three patients were considered to have nosocomial infections. The outcome of patients with bacteraemia showed an overall 14-day mortality of 47%.

The outcome of our patients seemed to be even worse compared to these data. As the isolates were broadly resistant to most antibiotic classes, various combination therapies were used with little effect. Although the isolates showed susceptibility to intermediate MICs to tigecycline as described previously [START_REF] Bratu | Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular epidemiology and in vitro activity of polymyxin B and other agents[END_REF], the clinical efficacy seemed to be low. One isolate became resistant to tigecycline (MIC >8 µg/ml) during therapy.

The source of the KPC-producing K. pneumoniae could not be fully elucidated, although two facts suggest that the strain has been imported from Greece: the isolation of a KPC-producing K. pneumoniae from a Greek patient with a PFGE pattern that was identical to most strains that have been isolated during the outbreak. In addition, the transposon structure, including the target site duplication sequence and a 100-bp deletion, was identical to two previously reported plasmids in K. pneumoniae isolates from France and Greece. Of interest, these plasmids were 80 kb in size, which are considerably smaller than the bla KPC-2 -harbour- In most incidents of isolation of KPC-producing K. pneumoniae in European countries, a link could be established to countries where occurrence of KPC had been reported previously. Naas et al. reported that the KPC-2 identified in France likely resulted from an intercontinental transfer of the KPC producer from the United States [START_REF] Naas | Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC in a Klebsiella pneumoniae isolate from France[END_REF], whereas at least one of the two recently described K. pneumoniae isolates with KPC-3 carbapenemase detected in the United Kingdom was probably imported into the United Kingdom from Israel [START_REF] Woodford | Arrival of Klebsiella pneumoniae producing KPC carbapenemase in the United Kingdom[END_REF]. Two KPC-2-producing K. pneumoniae isolates were exported from Greece; one to France [START_REF] Cuzon | Plasmidmediated carbapenem-hydrolyzing beta-lactamase KPC-2 in Klebsiella pneumoniae isolate from Greece[END_REF] and the other to Sweden [START_REF] Wisell | Identification of Klebsiella pneumoniae carbapenemase in Sweden[END_REF].

As the rapid spread of KPC has been reported from the United States [START_REF] Queenan | Carbapenemases: the versatile βlactamases[END_REF] and Israel [START_REF] Leavitt | Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae strains in an Israeli hospital[END_REF], we hoped to contain further spreading of KPC carbapenemase. A common source for the outbreak could not be established and the increasing risk of transmission with increasing contact times suggests that transmission via the hands of health care workers was the most likely mechanism of spread. With regard to the high pathogenicity of the organisms and the reported experiences with prolonged outbreaks [START_REF] Bratu | Emergence of KPCpossessing Klebsiella pneumoniae in Brooklyn, New York: epidemiology and recommendations for detection[END_REF][START_REF] Woodford | Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center[END_REF][START_REF] Leavitt | Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae strains in an Israeli hospital[END_REF], we decided to implement strict isolation of the cases for the whole period of hospitalisation.

In addition, we searched for asymptomatic carriers using two prevalence screenings in our institutions. Because no screening method was established in our laboratory, we decided to rely on a non-selective medium and a medium selective for ESBLs. We decided to test swabs from the throat and wounds, and rectal swabs, because these body sites were frequently positive in cases. The sensitivity of swabs taken from different body sites to detect carriers of KPC-producing K. pneumoniae has not been described so far. Thus, we cannot assess the risk of missing carriers. However, all known carriers who were tested by this screening method had positive results.

Although we may have terminated the outbreak in our institution, the high number of contact patients that resulted from long hospitalisation times and frequent transferring of the highly care-dependant patients makes it unlikely that the establishment of KPC in Germany could be prevented.

MICs derived by the Vitek 2 system were usually comparably low for imipenem and meropenem, whereas testing with microbroth dilution revealed higher MICs. This problem has been reported previously [START_REF] Tenover | Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing[END_REF]. However, the Vitek 2 system is widely used in German laboratories and, if included in the testing panel, ertapenem may serve as an indicator for the detection of KPC carbapenemases. The low in vitro efficacy of ertapenem in comparison to other carbapenems remains to be elucidated.

In conclusion, KPC-producing K. pneumoniae has been introduced to our university hospital, possibly from Greece, and was probably further transmitted by the hands of health care workers. The high number of contact patients to whom transmission may have occurred makes it likely that the KPC enzyme has established in Germany and probably in Europe. Thus, suspicion must be high in multi-resistant enterobacterial isolates with elevated MICs to ertapenem.

Potential conflicts of interest C.W. is a speaker for Pfizer and BD Diagnostics; M.A.W. is a speaker for Wyeth, Pfizer, MSD, Essex, Gilead, Astra Zeneca and Astellas. All other authors have no conflict of interest to declare.

Fig. 1

 1 Fig. 1 Pulsed-field gel electrophoresis (PFGE) of speI-digested DNA of Klebsiella pneumoniae. Lanes 2-4: unrelated non-K. pneumoniae carbapenemases (KPC)-producing isolates from three patients a, b, c; lanes 6-15: KPC-producing isolates from case patients 1 to 9; lanes 1 and 5: size markers are concatemers of phage λ DNA (multimers of 48.5 kb)

Fig. 2

 2 Fig. 2 Agarose gel of plasmid preparations from bla KPC-2 -positive isolates. From left to right: marker, K. pneumoniae patient 1, E. coli patient 1, E. coli J53 bearing pMG252

Table 1

 1 Characteristics of the case patients

	Patient	Age	Underlying	Length of stay	Detection of KPC-	Infection	Therapy	Outcome
		(years)	disease	(days)		producing K. pneumoniae	due to
								KPC-KP
				Before	Total Initial	Other culture-
				detection		detection	positive sites
				of KPC-
				KP
	1:	55	Urethral stenosis	55	78	Wound	Tracheal	Pneumonia	Tigecycline,	Deceased
	female		after radiation				secretion		colistin
			therapy
	2: male	42	Multiple trauma	11	29	Tracheal	Throat	-	-	Transferred
						secretion
	3:	71	Kidney	86	119	Tracheal	Wound, CVC,	Wound	Tigecycline,	Deceased
	female		transplantation,			secretion	urine,	infection,	colistin
			colon				throat,	pneumonia
			perforation				rectal
	4:	68	Arterial	53	129	Rectal	-	-	-	Transferred
	female		obstructive
			disease, above-
			knee amputation
	5:	55	Liver	66	157	Rectal	Urine	-	-	Transferred
	female		transplantation
	6: male	72	Oesophageal	147	140	Urine	-	-	-	Transferred
			cancer
	7:	67	Recurrent	28	33	Wound	-	-	-	Transferred,
	female		ovarian							deceased
			carcinoma
	8:	52	Obesity, gastric	262	316	Urine	Tracheal	Wound	Tigecycline,	Deceased
	female		sleeve operation				secretion,	infection,	meropenem,
							BAL, throat,	pneumonia,	fosfomycin,
							wound,	sepsis	gentamicin,
							rectal		colistin
	9: male	67	Rectal cancer	3	27	Wound	-	Abscess	Gentamicin,
									tigecycline

Discharged

KPC-KP=Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae; BAL = bronchoalveolar lavage

Table 2

 2 Antibiotic susceptibilities minimum inhibitory concentrations [MICs], mg/L) of KPC-2-producing Klebsiella pneumoniae and Escherichia coli isolates as determined by the Vitek 2 System

		Patient
		1		2	3	4	5		6	7	8	9
	Antibiotic	K. pneumoniae	E. coli				Isolate 1	Isolate 2
	Ertapenem	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8
	Imipenem	4	2	4	4	≥1 6	2	4	4	8	4	4
	Meropenem	2	1	2	2	8	≥16	≥2	2	2	1	2
	Ampicillin	≥32	≥32	≥32	≥32	≥32	≥32	≥32	≥32	≥32	≥32	≥32
	Cefotaxime	16	4	16	16	≥64	16	16	≥64	16	16	16
	Ceftazidime	≥64	4	≥64	≥64	≥64	≥64	≥64	≥64	≥64	≥64	≥64
	Cefpodoxime	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8
	Piperacillin	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128
	Piperacillin/tazobactam	≥128	64	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128
	Ciprofloxacin	≥4	≥4	≥4	≥4	≥4	≥4	≥4	≥4	≥4	≥4	≥4
	Moxifloxacin	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8	≥8
	Gentamicin	4	≥1 6	4	4	4	4	4	4	2	4	4
	Tobramycin	≥16	8	≥16	≥16	≥16	≥16	≥16	≥16	≥16	≥16	≥16
	Tetracycline	4	≥1 6	4	4	4	8	8	4	4	4	4
	Tigecycline	2	≤0 . 5	2	2	2	4	4	1	2	2	2
	Cotrimoxazole

Table 3

 3 Antimicrobial susceptibility determined for donor, recipient and transconjugant by microbroth dilutiontesting according to the NCCLS/ CLSI

	Antibiotic	Recipient, donor and transconjugant strains
		E. coli J 53	Patient 1 K. pneumoniae	conj. #104	Patient 1 E. coli	conj. #99
	Amoxicillin	8	>256	>256	>256	>256
	Amoxicillin+CLA	8	>256	256	>256	>256
	Piperacillin	2	>128	>128	>128	>128
	Piperacillin+TZB	2	>256	>256	>256	256
	Cefoxitin	4	128	32	32	16
	Cefotaxime	0.12	>256	>256	>256	>256
	Cefotaxime+CLA	0.06	16	4	16	4
	Ceftazidime	0.5	>128	32	32	32
	Ceftazidime+CLA	0.25	128	4	32	4
	Cefepime	0.06	>256	16	>256	64
	Imipenem	0.25	128	16	32	16
	Imipenem+CLA	0.125	16	2	8	1
	Meropenem	0..03	32	8	16	8
	Meropenem+CLA	<0.015	8	0.125	8	0.5
	Ertapenem	<0.008	64	8	32	8
	Ertapenem+CLA	<0.008	32	4	8	2
	Ciprofloxacin	<0.015	128	<0.015	128	<0.015
	Gentamicin	0.25	2	0.25	0.25	0.25
	Tetracycline	1	4	1	256	256
	Trimethoprim-sulfamethoxazole	0.03/0.57	128/2432	0.06/1.14	128/2432	128/2432
	CLA = clavulanic acid; TZB = tazobactam

Eur J Clin Microbiol Infect Dis (2010) 29:563-570


Margarida B Prata

Solange I Mussatto
email: solange@deb.uminho.pt

• Lı

R Rodrigues

Jose ´a Teixeira

Á S I Mussatto

L R Rodrigues

J A Teixeira

Fructooligosaccharide production by Penicillium expansum

Keywords: b-fructofuranosidases, Fructooligosaccharides, Penicillium expansum

Fructooligosaccharides (FOS) production by Penicillium expansum was evaluated. In a first stage, the best conditions for P. expansum growth and sporulation were established with potato/dextrose/ agar being the most suitable medium at between 22 and 25°C, giving good growth and good sporulation. The inocula from this medium were used for FOS production using shake-flask cultures, and yielded 0.58 g FOS/g sucrose (3.25 g FOS/l.h), demonstrating the potential of this strain for sucrose conversion to FOS.

Introduction

Fructooligosaccharides (FOS) are non-conventional sugars that cannot be hydrolyzed by gastrointestinal enzymes. They have a low caloric value and can promote beneficial effects to the host through the selective stimulation of indigenous bacteria like bifidobacteria and lactobacilli (Mussatto and Mancilha 2007;Teitelbaum and Walker 2002). They are formed by b(2 ? 1)-linked fructose (F) units with a last one, a(2 ? 1), attached to a terminal glucose (G) moiety. These oligomers are mainly composed of 1-kestose (GF 2 ), 1-nystose (GF 3 ), and 1-b-fructofuranosyl nystose (GF 4 ), which are found in many fruits and vegetables, although in too low a concentration to exert any beneficial effect (Yun 1996). Commercially, FOS can be produced by from sucrose using microbial b-fructosyltransferases (FTases) or b-fructofuranosidases (FFases) with high transfructosylating activity (Chen and Liu 1996). Such enzymes can be obtained from plants, bacteria or fungi, with the latter giving the greatest yields (Yun 1996;Antosova ´et al. 2008). Several strains, especially those from the Aspergillus genus, are good FOS producers (Sangeetha et al. 2005). However, there is no report on FOS production by Penicillium expansum. The present work, therefore, has evaluated FOS production by this fungus. Initially, the best conditions for producing an inoculum of fungal spores were established and this was then followed by an examination of FOS production in shake-flask culture.

Materials and methods

Microorganism and culture conditions

Penicillium expansum MUM 02.14 was obtained from Micoteca of the Centre of Biological Engineering, University of Minho, Portugal. Mycelia growth and sporulation at 22 and 27°C, in potato/dextrose/agar (PDA) and Czapek-Dox agar (CZ) media, were evaluated, in Petri dish culture. Plates were inoculated with 40 ll spore suspension containing 7.5 9 10 7 spores/ml. Six plates (3 PDA and 3 CZ) were incubated at 22°C and six similar ones at 27°C. Fermentation medium and conditions for FOS production FOS production was carried out in 500 ml Erlenmeyer flasks containing 100 ml culture medium (% w/v): sucrose (20), yeast extract (2.75), NaNO 3 (0.2), K 2 HPO 4 (0.5), MgSO 4 9 7H 2 O (0.05), and KCl (0.05), sterilized at 112°C for 15 min. The flasks were inoculated with 1 ml spore suspension containing around 1.8 9 10 7 spores/ml, and incubated at 25°C with shaking at 160 rpm for 48 h. Experiments were carried out in duplicate; and samples were aseptically collected for determining FOS (1-kestose, 1-nystose, and 1-b-fructofuranosyl nystose) and residual sugars (sucrose, fructose and glucose) concentrations, along with extracellular b-fructofuranosidase activity. Biomass concentration was determined at the end.

Analytical methodology

Microorganism growth and sporulation

Penicillium expansum growth in Petri plates was assessed daily for 7 days. Sporulation was determined by counting the spores in a Neubauer chamber after suspending them in 5 ml 0.1% (w/v) Tween 80. Biomass in the fermentation medium (g/l) was determined after drying at 105°C.

Sugars and fructooligosaccharides determination

FOS and residual sugar concentration in the fermentation broth were determined by HPLC (Mussatto et al. 2009). FOS yield was calculated as the proportion of the sum of 1-kestose, 1-nystose and 1-b-fructofuranosyl nystose to the initial sucrose concentration (Y P/St in g/g), and to consumed sucrose concentration (Y P/Sc in g/g). FOS productivity (Q P in g/l.h) was calculated as the total FOS production (g/l) by fermentation time (h).

Enzyme activity

Samples of the filtered fermentation broth were used as extracellular enzyme source. The b-fructofuranosidase (FFase) activity was determined by measuring the amount of glucose produced from sucrose (Yoshikawa et al. 2006).

Statistical analysis

The results were analyzed by the Tukey test using the software Statgraphics version 4.1. A P value less than 0.05 indicated significant differences among samples.

Results and discussion

Growth and sporulation conditions for Penicillium expansum Penicillium expansum grew well when at 22 and 27°C in both PDA and CZ media; however, the sporulation occurred only on PDA medium (as shown in Supplementary Figs. 1 and2). At the same temperature, there was no statistically significant differences between growth in the two media (Table 1). Nonetheless, the growth was greater at 22°C. On the other hand, sporulation was statistically different when varying the culture medium and temperature: CZ was less suitable than PDA for sporulation at both temperatures. Such results can be explained by the low N concentration in PDA medium (Supplementary Table 1).

In a final step, fungal growth and sporulation was also evaluated at 25°C which was similar to that at 22°C. Therefore, PDA is suitable for good growth and sporulation of P. expansum between 22 and 25°C.

Fructooligosaccharides production

Figure 1a shows the time course of sucrose consumption and FOS production by P. expansum grown at the previously established cultivation conditions.

There was an initial lag phase up to 12 h before FOS formation began. Between 12 and 24 h, sucrose conversion to FOS commenced and in the following 12 h, the process attained the maximum FOS production (117.7 g/l) with almost total depletion of sucrose. Maximum production of FOS corresponded to a final product containing 1-kestose (GF 2 -80%), 1-nystose (GF 3 -19%), and 1-b-fructofuranosyl nystose (GF 4 -1%) (Fig. 1b). According to Cruz et al. (1998) FOS synthesis always occurs in the sequence GF ? GF 2 ? GF 3 ? GF 4 , as a consequence of the increasing Km values for such products presented by the transfructosylase. Thus, high concentrations of the preceding oligosaccharide are necessary for the synthesis of the homolog with one more fructose unit. After 36 h, due to sucrose exhaustion from the media, the microorganism started consuming the produced FOS and, therefore, the total FOS concentration decreased.

FOS production by P. expansum occurred with good yield (Y P/St = 0.58 g/g; Y P/Sc = 0.68 g/g) and productivity (Q P = 3.25 g/l.h) when compared to the results obtained for other producer strains like Aspergillus japonicus (Y P/St = 0.61 g/g, Y P/Sc = 0.64 g/g, Q P = 5.36 g/l.h) (Mussatto et al. 2009), being similar to the maximum yield normally found for microorganisms (55-60%, w/w) (Yun 1996;Nishizawa et al. 2001). Nevertheless, the values reported here can probably be improved by establishing the best conditions for performing this fermentative process.

FFase activity, responsible for sucrose conversion to FOS, increased throughout the fermentation, reaching a maximum value of 41 U/ml at the end (Fig. 2). Such a value compares well with other activities for FOS producers such as A. japonicus * Each value was calculated as the overall average of colony diameter measurements (in 3 different directions) taken every day, during 7 days, for each of the three replicates ** Same letters denotes no statistically significant differences at 95% confidence level In conclusion, P. expansum has potential for sucrose conversion to FOS. The present results can probably be improved by establishing the best conditions for the fermentation process. Nevertheless, since this is the first study on the FOS production by P. expansum, the obtained results open up possibilities to develop an efficient process for producing FOS with yields higher than that usually found in batch processes (55-60%, w/w) (Yun 1996), which is the main challenge for the FOS industries nowadays.

Fig. 1

 1 Fig. 1 Kinetic behavior of fructooligosaccharides production by Penicillium expansum using shake-flask cultures: (a) sucrose consumption and fructooligosaccharides production (as the sum of 1-kestose, 1-nystose, and 1-b-fructofuranosyl nystose); (b) 1-kestose, 1-nystose, and 1-b-fructofuranosyl nystose production

Fig. 2

 2 Fig. 2 Activity of b-fructofuranosidase during fructooligosaccharide production from sucrose by Penicillium expansum using shake-flask cultures

Table 1

 1 Multiple comparison analysis of mycelial growth rate and sporulation results of Penicillium expansum cultivated in Potato Dextrose Agar (PDA) and Czapek-Dox Agar (CZ), at 22 and 278C

	Culture media/temperature (8C)	Radial growth rate (mm/h)*	Sporulation (10 7 spores/ml)
		Average**	Average**
	CZ/27	0.157 a	0 a
	PDA/27	0.148 a	14 b
	CZ/22	0.242 b	3.4 a
	PDA/22	0.249 b	29 c

Acknowledgement The financial support from FCT, the Portuguese Foundation for Science and Technology (research project SFRH/BPD/38212/2007) is gratefully acknowledged. References Antosova ´M, Illeova ´V, Vanda ´kova ´M, Druzkovska ´A, Polakovic M (2008) Chromatographic separation and kinetic properties of fructosyltransferase from Aureobasidium pullulans. J Biotechnol 135:58-63 Chen WC, Liu CH (1996) Production of b-fructofuranosidase by Aspergillus japonicus. Enzyme Microb Tech 18:153-160 Cruz R, Cruz VD, Belini MZ, Belote JG, Vieira CR (1998) Production of fructooligosaccharides by the mycelia of Aspergillus japonicus immobilized in calcium alginate. Bioresour Technol 65:139-143 Mussatto SI, Mancilha IM (2007) Non-digestible oligosaccharides: a review. Carbohyd Polym 68:587-597 Mussatto SI, Aguilar CN, Rodrigues LR, Teixeira JA (2009) Fructooligosaccharides and b-fructofuranosidase production by Aspergillus japonicus immobilized on lignocellulosic materials. J Mol Catal B-Enzym 59:76-81 Nishizawa K, Nakajima M, Nabetani H (2001) Kinetic study on transfructosylation by beta-fructofuranosidase from Aspergillus niger ATCC 20611 and availability of a membrane reactor for fructooligosaccharide production. Food Sci Technol Res 7:39-44 Sangeetha PT, Ramesh MN, Prapulla SG (2005) Recent trends in the microbial production, analysis and application of fructooligosaccharides. Trends Food Sci Tech 16:442-457 Teitelbaum JE, Walker WA (2002) Nutritional impact of preand probiotics as protective gastrointestinal organisms. Annu Rev Nutr 22:107-138 ul-Haq I, Ali S, Aslam A, Qadeer MA (2008) Biosyntheses of L-DOPA by Aspergillus oryzae. Bioresour Technol 99: 7-12 Wang L-M, Zhou H-M (2006) Isolation and identification of a novel Aspergillus japonicus JN19 producing -fructofuranosidase and characterization of the enzyme. J Food Biochem 30:641-658 Yoshikawa J, Amachi S, Shinoyama H, Fujii T (2006) Multiple b-fructofuranosidases by Aureobasidium pullulans DSM2404 and their roles in fructooligosaccharide production. FEMS Microbiol Lett 265:159-163 Yun JW (1996) Fructooligosaccharides-occurrence, preparation, and application. Enzyme Microb Tech 19:107-117


Luo Liu

Rolf D Schmid

Vlada B Urlacher
email: vlada.urlacher@itb.uni-stuttgart.de

Á R D Schmid

Engineering cytochrome P450 monooxygenase CYP 116B3 for high dealkylation activity

Keywords: Cytochrome P450, Dealkylation, Directed evolution, High-throughput screening

Cytochrome P450 monooxygenase CYP-116B3 from Rhodococcus ruber catalyzes the dealkylation of 7-ethoxycoumarin and the hydroxylation of substituted and unsubstituted aromatics. However, since activities were quite low, a combination of site-specific mutagenesis and directed evolution was applied to produce 7800 variants of CYP116B3, which were screened via a newly developed highthroughput screening system based on the dealkylation of 7-ethoxycoumarin catalyzed by recombinant E. coli. The best mutant was found after four rounds of directed evolution and had a 240-fold increased deethylation activity toward 7-ethoxycoumarin (223 nmol product/nmol P450.min) and a 10-fold increased demethylation activity toward 7-methoxycoumarin (9 nmol product/nmol P450.min).

Introduction

The cytochrome P450 enzymes (P450s or CYPs) constitute a large super-family of heme-thiolate proteins currently comprising well over 11500 distinct sequences from all phyla including animals, plants, fungi, and bacteria (http://drnelson.utmem.edu/cyto chromeP450.html) [START_REF] Nelson | The cytochrome P450 homepage[END_REF]. Cytochrome P450s catalyze monooxygenase reactions that introduces O 2 into generally non-polar, aromatic or aliphatic molecules, thereby leading to hydroxylation, dealkylation, aromatization, epoxidation or cleavage of carboncarbon bonds [START_REF] Mansuy | Cytochromes P450 and model systemsgreat diversity of catalyzed reactions[END_REF][START_REF] Sono | Hemecontaining oxygenases[END_REF][START_REF] Cryle | Reactions catalyzed by bacterial cytochromes P450[END_REF]. Because of their unique and versatile catalytic properties, P450s are of interest as potential biocatalysts for industrial applications. However, most P450s display rather low activity and stability and therefore need to be optimized to fulfil the technical requirements [START_REF] Urlacher | Cytochrome P450 monooxygenases: perspectives for synthetic application[END_REF].

Site-specific mutagenesis or directed evolutionas well as a combination of both-can be applied to improve or change enzyme features. A great advantage of directed evolution techniques is that they can be applied without structural knowledge of a protein [START_REF] Arnold | Directed evolution library creation: methods and protocols[END_REF]. Methods of directed evolution alone or in combination with site-specific mutagenesis have successfully been used to engineer bacterial P450s. For example, the self-sufficient fatty acid hydroxylase P450 BM-3 from Bacillus megaterium [START_REF] Munro | P450BM3: the very model of a modern flavocytochrome[END_REF] was engineered for oxidation of n-alkanes [START_REF] Farinas | Directed evolution of a cytochrome P450 monooxygenase for alkane oxidation[END_REF], terpenoids (Urlacher et al. 2006) and human metabolites [START_REF] Otey | Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450[END_REF]. Furthermore, using error-prone PCR the stability of this enzyme in the presence of organic solvents was improved significantly [START_REF] Wong | Laboratory evolution of cytochrome P450BM-3 monooxygenase for organic cosolvents[END_REF]).

The results of those studies demonstrate that the success of directed evolution experiments is often depended on the efficiency of the screening methods applied, since a large number of variants is usually generated and must be tested [START_REF] Olsen | High-throughput screening of enzyme libraries[END_REF].

Previously, we isolated and characterized CYP-116B3 from Rhodococcus ruber DSM 44319 [START_REF] Liu | Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319[END_REF]). The enzyme is a self-sufficient fusion protein consisting of a heme-containing P450 domain, a FMN-containing reductase and a 2Fe-2S-ferredoxin. CYP116B3. It catalyzes the dealkylation of 7-ethoxycoumarin and the hydroxylation of several substituted and unsubstituted aromatic hydrocarbons in the presence of NADPH. However, the activity was low in all cases. A turnover rate of 0.9 nmol/nmol P450.min was determined for the dealkylation of 7-ethoxycoumarin by wild-type CYP116B3, which is one order of magnitude lower compared to the activity of P450 BM-3 toward its physiological substrates fatty acids [START_REF] Munro | P450BM3: the very model of a modern flavocytochrome[END_REF].

To improve the activity of CYP116B3, we first applied site-specific mutagenesis followed by four rounds of directed evolution. The best enzyme found was a quintuple mutant, which demonstrated a 240fold increased deethylation activity toward 7-ethoxycoumarin and a 10-fold increased demethylation activity toward 7-methoxycoumarin.

Materials and methods

Materials and stains

7-Ethoxycoumarin, 7-methoxycoumarin, and 7-hydroxycoumarin were purchased from Sigma-Aldrich (Germany). All other chemicals were purchased from Fluka (Switzerland).

Site-specific mutagenesis

The structure of the heme domain of CYP116B3 was modeled using the program Modeller 8v2 (http:// salilab.org/index.html). The replacement of alanine at position 109 by phenylalanine was performed using the Quick-Change Kit (Stratagene) as recommended by the manufacturer. The plasmid pET28a(?)_ CYP116B3 constructed previously [START_REF] Liu | Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319[END_REF] was used as template. The primers for PCR were designed as follows: 5 0 -CGACTACGCCATGTTCC GGACCCTCGTGAACG-3 0 and 5 0 -CGTTCACGA GGGTCCGGAACATGGCGTAGTCG-3 0 . The mutation sites are underlined. All mutants were expressed in E. coli BL21(DE3) (Novagen, UK) and characterized as previously described [START_REF] Liu | Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319[END_REF].

Error-prone PCR The E. coli NovaBlue(DE3) strain (Novagen, UK) was used as an expression host. Random libraries were generated by error-prone PCR using the plasmid pET28a(?) harboring the CYP116B3_A109F gene as template. Mutagenesis was performed only on the heme domain. The following primers were used: 5 0 -TAATACGACTCACTATAGGG-3 0 and 5 0 -TGT TCGTGGACGTAGCGCGA-3 0 . Taq-polymerase was chosen because of its high natural error rate. The error-prone PCR reaction mixtures contained 1.5 mM MgCl 2 for stabilization of non-complementary pairs. Unequal dNTPs concentrations, 0.05 mM MnCl 2 and 0.05 mM dITP were applied to increase the error rate during PCR.

Primary screening for enzyme activity on a multiwell plate scale

Expression of the CYP116B3 wild-type enzyme and mutants in E. coli NovaBlue(DE3) was performed in deep-well plates (Greiner Bio-One GmbH, Germany). Each well, containing 600 ll LB-medium supplemented with 50 mg kanamycin/l and 5 mg thiamine/l was inoculated with a single colony. Cells were grown at 37°C, 200 rpm for about 4.5 h to an OD 590 of *0.35. P450 expression was induced by addition of 0.4 mM IPTG. The cells were cultivated further for 24 h at 30°C, 180 rpm. The plates were centrifuged at 4,0009g/w, 4°C for 30 min. The supernatants were discarded and cell pellets resuspended in 190 ll 50 mM potassium phosphate buffer (pH 7.5) and transferred into black 96-well plates FluoroNunc (Sigma-Aldrich). Each well was supplemented with 10 ll 25 mM 7-ethoxycoumarin (dissolved in DMSO). The 96-well plates were immediately put into a multiwell plate reader (FLUOstar, BMG) and the fluorescence intensity of the product, 7-hydroxycoumarin (excitation at 405 nm, emission at 460 nm), was measured. The measurements were carried out for nine cycles with 90 s intervals.

Secondary screening

To sort out false positive clones, the best candidate mutants were expressed in shaking flasks containing 100 ml LB medium supplemented with 50 mg kanamycin/l. Expression and enzyme activity measurements were performed as previously described [START_REF] Liu | Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319[END_REF]. Concentrations of CYP116B3 variants were calculated from CO-difference spectra as described elsewhere [START_REF] Omura | The carbon monoxide-binding pigment of liver microsomes[END_REF]. Finally the best variants were validated by automated DNA sequencing (GATC Biotech, Konstanz, Germany). All experiments were performed at least three times in duplicate.

Results and discussion

Since the crystal structure of CYP116B3 has not yet been solved, a homology model of the heme domain was created based on sequence alignments and crystal structures of P450 terp (CYP108A1) (PDB ID, 1CPT), P450 epoK (CYP167A1) (PDB ID, 1PKF), and P450 eryF (CYP107A1) (PDB ID1Z8O). Examining the homology model revealed a small region at the ceiling of the substrate binding pocket which might control enzyme activity. Since the alanine at position 109 within this region is located directly above the heme group, we expected that introduction of the bulky amino acid phenylalanine at this position would facilitate better stabilization of smaller molecules and thus result in enhanced activity and/or regioselectivity. Indeed, the single mutant CYP116B3 A109F demonstrated nearly 3-times higher activity toward 7-ethoxycoumarin and PAHs (naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene and ethyl benzene) compared to the wild-type enzyme. However, the substrate specificity and regioselectivity of this mutant remained unchanged.

As directed evolution does not require any structural information for construction of a random mutant library, we decided to apply this approach for further improvement of CYP116B3. A random mutant library was generated by error-prone PCR. The most critical step during directed evolution is the development of a simple, reproducible and fast high-throughput screening (HTS) for the direct measurement of enzyme activity. Since P450s depend on the delivery of electrons, which are in most cases ultimately delivered from NAD(P)H, it is possible to follow NAD(P)H oxidation at 340 nm. However, the enzyme/substrate match is often sub-optimal and results in reducing equivalents from NAD(P)H being channeled away from product formation to give instead hydrogen peroxide and water (so called ''uncoupling''). NAD(P)H consumption and product formation are therefore not always correlated. More accurate and precise methods are based on direct measurements of substrate consumption or product formation. Fortunately 7-hydroxycoumarin is fluorescent and its formation can be monitored directly. Therefore this method was used for activity screenings within this study.

To avoid the tedious cell lysis and high costs for the co-factor NADPH, the activity screening was carried out with resting E. coli cells in 96-well plates. Expression of CYP116B3 in LB-medium showed better results compared to those achieved with TBmedium. Interestingly, the addition of a heme precursor, 5-aminolevulinic did not affect the expression level of CYP116B3.

The first set of experiments was performed with 1.2 mg resting cells (cell wet wt) expressing wildtype CYP116B3 to investigate if the dealkylation activity of the enzyme could be measured with recombinant E. coli. Effect of the cell permeabilizer polymyxin B was also investigated. E. coli containing the plasmid pET28a(?) (as purchased from the manufacturer) was used as a negative control. A positive control was performed with cell lysates containing CYP116B3 isolated from an equivalent amount of recombinant E. coli. Utilizing this set-up, dealkylation activity of CYP166B3 could be measured with whole E. coli cells and addition of the permeabilizer, polymyxin B, did not affect the screening results (Table 1).

The single mutant A109F was used as template for the first round of error-prone PCR. Statistical analysis of the results gained in the primary screening revealed that 35% of the generated clones were inactivated through mutagenesis. Since enzyme activity in the whole-cell assay could not be normalized to the enzyme concentration, it was not possible to distinguish between false positive clones (originating from high amounts of cells and/or higher expression of CYP116B3) from clones with enhanced specific activity. Consequently, the most active clones yielding double at least the fluorescence response of A109F were applied to a secondary screening.

The secondary screening was performed in shaking flasks as described in the ''Materials and methods'' section. Concentrations of CYP116B3 variants in the soluble protein fractions after cell lysis were calculated and the specific activities toward 7-ethoxycoumarin were determined (Table 2). Only three out of 12 variants exhibited increased specific activity, whereas other ''positive'' variants were assigned as false positives. The variant 4E01 had the highest specific activity but was poorly stable under the expression conditions applied. We decided to exclude this variant and continue our experiments with variant 7F08 that demonstrated high activity and stability upon expression. DNA-sequencing of variant 7F08 revealed that isoleucine at position 267 was replaced by leucine. This double CYP116B3 mutant A109F/I267L was used as template for the second round of error-prone PCR.

Three additional rounds of directed evolution were carried out according to the procedure described above, and 7800 variants were screened in total. After the fourth round of error-prone PCR, the activity toward 7-ethoxycoumarin could be increased from 2.6 nmol/nmol P450.min (measured with the single mutant A109F) to 141 nmol/nmol P450.min (variant 74H10, T91S/A109L/I179F/I267L) and to 223 nmol/ nmol P450.min (variant 70A08, A86T/T91S/A109F/ I179F/I267L) (Table 3). Interestingly, phenylalanine at position 109 was substituted by leucine in variant 74H10, which is the position that was originally chosen for site-specific mutagenesis.

We also determined the activity of the two best mutants, 74H10 and 70A08, toward coumarin and its derivative 7-methoxycoumarin. Both mutants accepted 7-methoxycoumarin as substrate and demonstrated increased specific activity up to 9 nmol/ nmol P450.min. However, no hydroxylation activity toward coumarin was observed. Furthermore, other substrates of wild-type CYP116B3 including naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene, and ethyl benzene were tested. However, neither the variant 70A08, nor 74H10 displayed enhanced activity toward any of those substrates.

Conclusions

Using site-specific mutagenesis at position 109 the dealkylation activity of CYP116B3 was increased 3-fold compared to the wild-type enzyme. A CYP-116B3 mutant library was generated by four rounds of error-prone PCR to further enhance the enzyme's activity. A newly developed high throughput screening utilizing resting recombinant E. coli cells was applied for library screening. Finally, two mutants, 70A08 (A86T/T91S/A109F/I179F/I267L) and 74H10 (T91S/A109L/I179F/I267L), were identified that exhibited 150-or 240-fold increased dealkylation activity. Several ''hotspot'' positions have been identified in the substrate binding pocket and on the surface Activity was measured with cell lysates as described in the ''Materials and methods'' section of CYP116B3, which can be applied to further sitespecific or saturation mutagenesis studies. Nevertheless, the correlation between the mutated positions and activity of the respective mutants will not be discussed herein, since a detailed protein structure is not available yet.

Table 1

 1 Activity of recombinant E. coli cells toward 7-ethoxycoumarin in the presence and absence of polymyxin B in a total reaction volume of 200 ll If polymyxin treatment was applied, 20 ll 0.6 mM polymyxin B (dissolved in 50 mM potassium buffer, pH 7.5) was added to 160 ll cell suspension. The positive control was represented by cell lysate derived from 1.2 mg E. coli cells. No activity was detected in the negative control with E. coli cell harboring pET28a(?)CWW cell wet weight a 10 ll of cell lysate derived from 1.2 mg CWW, contain 0.18 lM CYP116B3

	Biocatalyst	Polymyxin B (lM)	Activity (lM/min)
	1.2 mg CWW	-	0.19
	1.2 mg CWW	60	0.18
	Cell lysate a	-	0.17

Table 2

 2 Dealkylation activity of 12 variants chosen from the primary screening

	Variants	Product formation rate
		(nmol/nmol P450.min)
	2A09	5.41
	4A08	1.27
	4A09	1.26
	4A10	1.37
	4E01	7.22
	4G10	1.48
	5F11	1.13
	7A10	1.61
	7E09	1.62
	7F08	6.67
	8A05	1.6
	8A10	1.7

BiotechnolLett (2010) 32:841-845

Acknowledgments We thank Marco Girhard and Clemens von Buhler for constructive discussions and critical review of the manuscript.