Olga Pérez-Moreno

Maria José Centelles-Serrano

Maria Cortell-Ortolá

Isabel Fort-Gallifa

Joaquim Ruiz

Maria Isabel Llovet-Lombarte

Ester Picó-Plana

Anna Maria Jardí-Baiges

Ester Picó- Plana

Molecular epidemiology and resistance mechanisms involved in reduced susceptibility to amoxicillin/clavulanic acid in isolates from a chronic care centre

Keywords:

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A c c e p t e d M a n u s c r i p t ABSTRACT The aim of this work was to investigate the molecular epidemiology and mechanisms responsible for reduced susceptibility to amoxicillin/clavulanic acid (AMC) among cefazolin-susceptible Klebsiella pneumoniae isolates from patients admitted to a chronic care institution. In total, 51 (29.8%) of 171 K. pneumoniae isolates recovered between 2006 and 2008 were non-susceptible to AMC, of which 45 were susceptible to cefazolin. Nucleotide sequencing analysis revealed that 19 produced IRT-11 and the remaining 26 were OXA-1-producers. All of the OXA-1-producing isolates harboured the aac(6')-Ib-cr-bla OXA-1 cassette array, which in 23 isolates was located together with catB3 and arr3 within a class1 integron and associated with qnrS2 (in 3 cases the integron lacked the qacE1 and sul1 or sul3 genes). Genotyping analysis performed by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) identified three different patterns among IRT-11-producing isolates (E1 to E3), with E1 being the most prevalent (63.2%), whilst the OXA-1producing isolates were assigned to patterns E3 and E3a (isolates carrying typical class 1 integrons), E4 (isolates carrying defective integrons) and E5 (isolates without integrons). Genes encoding IRT-11 and OXA-1 were transferred by conjugation, and aac(6')-Ib-cr and qnrS2 were systematically co-transferred with bla OXA-1 . These results demonstrate that the high prevalence of decreased susceptibility to AMC among K. pneumoniae isolates from a chronic care hospital was mainly due to the simultaneous spread of two different clones, one of which comprised isolates producing IRT-11 and the other one comprised isolates that had acquired either the bla OXA-1 gene located in a class 1 integron and linked to qnrS2 or the bla IRT-11 gene.

A c c e p t e d M a n u s c r i p t

INTRODUCTION

Klebsiella pneumoniae is a Gram-negative bacterium that is present as a saprophyte in the nasopharynx and intestinal tract of humans at rates of 1-6% and 5-38%, respectively. However, these colonisation rates increase dramatically in hospitalised patients in direct proportion to the length of stay and antibiotic use. Moreover, K.

pneumoniae is very well adapted to the hospital environment since it exhibits higher survivability on hands and environmental surfaces than other Enterobacteriaceae and the wherewithal to spread rapidly and provoke nosocomial outbreaks [START_REF] Podschun | Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors[END_REF].

Klebsiella pneumoniae is naturally resistant to amino-and carboxy-penicillins owing to the production of a chromosomally encoded SHV-1-like -lactamase. Most strains of K. pneumoniae are susceptible to narrow-spectrum cephalosporins and to associations of -lactams with -lactamase inhibitors (BLIs). Hyperproduction of SHV-1 is the main mechanism responsible for resistance to these compounds in K. pneumoniae, whilst production of plasmid-borne enzymes conferring resistance to BLIs but not to narrow-spectrum cephalosporins, such as inhibitor-resistant TEM (IRT)-type or SHV-type -lactamases, is uncommon in this species [START_REF] Lemozy | First characterization of inhibitor-resistant TEM (IRT) -lactamases in Klebsiella pneumoniae strains[END_REF][START_REF] Dubois | Molecular and biochemical characterization of SHV-56, a novel inhibitor-resistant -lactamase from Klebsiella pneumoniae[END_REF]. On the other hand, simultaneous production of OXA-1 -lactamase (another enzyme poorly inhibited by clavulanic acid, tazobactam or sulbactam and with negligible activity against first-generation cephalosporins) and the cr variant of Acc(6')-Ib has recently been reported among K. pneumoniae isolates producing either plasmid-encoded cephamycinases linked to complex class 1 integrons and to qnr genes [START_REF] Quiroga | Complex class 1 integrons with diverse variable regions, including aac(6')-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial isolates from Argentina[END_REF] or extended-spectrum -lactamases (ESBLs) [START_REF] Machado | Portuguese Resistance Study Group. Dissemination in Portugal of CTX-M-15-, OXA-1-, and TEM-1-producing Enterobacteriaceae strains containing the aac(6')-Ib-cr gene, which encodes an aminoglycoside-and fluoroquinolone-modifying enzyme[END_REF].

A c c e p t e d M a n u s c r i p t

A retrospective survey of antibiotic resistance carried out in the microbiology laboratory of Hospital Verge de la Cinta (Tortosa, Catalonia, Spain) in 2005 revealed a high rate of decreased susceptibility to amoxicillin/clavulanic acid (AMC) among cefazolin-susceptible K. pneumoniae isolates recovered from patients from a chronic care hospital since 2003. The aim of this study was to follow the evolution of K. pneumoniae isolates displaying the former -lactam resistance phenotype and to investigate their molecular epidemiology and the underlying resistance mechanisms.

Material and methods

Bacterial isolates

All K. pneumoniae isolates with decreased susceptibility to AMC [minimum inhibitory concentration (MIC) >8/4 mg/L], recovered from clinical samples from patients admitted to a chronic care centre for whom the microbiology laboratory of the Hospital Verge de la Cinta provides its services, were prospectively collected between January 2006 and December 2008. Only one isolate per patient was considered.

Antimicrobial susceptibility

In vitro susceptibility to amoxicillin, AMC, piperacillin/tazobactam (TZP), cefazolin, cefoxitin, cefuroxime, ceftazidime, cefotaxime, cefepime, imipenem, meropenem, nitrofurantoin, fosfomycin, tetracycline, chloramphenicol, sulfamethoxazole/trimethoprim, nalidixic acid, ciprofloxacin, gentamicin, kanamycin and tobramycin was studied using the Wider commercial microdilution system (Francisco Soria Melguizo, Madrid, Spain) and to kanamycin, streptomycin, A c c e p t e d M a n u s c r i p t spectinomycin and sulfonamides by disk diffusion following the guidelines and applying the criteria for susceptibility and resistance of the Clinical and Laboratory Standards Institute (CLSI) [START_REF]Performance standards for antimicrobial susceptibility testing; nineteenth informational supplement[END_REF]. AMC was tested at a 2:1 ratio and TZP was assayed at a fixed inhibitor concentration of 4 mg/L.

Detection of resistance genes and class 1 integrons

Polymerase chain reaction (PCR) amplification followed by nucleotide sequencing was performed under conditions and with primers previously described (Table 1) to screen for genes encoding resistance to -lactams (bla TEM , bla SHV-1 , bla OXA-1 and bla PSE-1 ), aminoglycosides [aac(6')-Ib] and sulfonamides (sul1 and sul3) and for the qnrA, qnrB and qnrS (plasmid-mediated quinolone resistance), qacE1 and orf513 (element associated with insertion sequence ISCR1) as well as to detect mutations in the quinolone-resistance determining regions (QRDRs) of gyrA and parC genes. The presence of class 1 integrons was investigated by PCR amplification using specific primers aimed to detect the intI1 gene (Table 1). Gene integron content within the variable region of class 1 integrons was assessed by PCR using 5'-conserved segment (5'-CS) and 3'-CS set of primers or by PCR mapping assays employing 5'-CS plus bla OXA-1 reverse primers and 3'-CS plus bla OXA-1 forward primers and subsequent nucleotide sequencing (Table 1). In isolates lacking the qacE1 gene, the arr3 reverse specific primer (Table 1) was used instead of 3'-CS.

Molecular epidemiological analysis

DNA fingerprinting was achieved by means of enterobacterial repetitive intergenic consensus (ERIC)-PCR using the ERIC-2 primer (5'-A c c e p t e d M a n u s c r i p t AAGTAAGTGACTGGGGTGAGCG-3') and repeating reactions at least twice in each isolate. Isolates were considered epidemiologically unrelated if their patterns differed by more than one band [START_REF] Deplano | Typing of Staphylococcus aureus and Staphylococcus epidermidis strains by PCR analysis of inter-IS256 spacer length polymorphisms[END_REF].

Conjugation assays

Conjugation experiments were carried out in Luria-Bertani broth with azide-resistant Escherichia coli J53 as the recipient strain. Transconjugants were selected on trypticase soy agar plates containing 150 mg/L sodium azide and 100 mg/L amoxicillin.

Results

-Lactamase characterisation and antibiotic susceptibility

In total, 51 (29.8%) of 171 K. pneumoniae isolates recovered from clinical samples of different patients admitted to the chronic care hospital over the 3-year study period showed decreased susceptibility to AMC, 6 of which were resistant to cefazolin and displayed a resistant phenotype consistent with the production of AmpC (four isolates) or ESBLs (two isolates), whilst the remaining 45 isolates, all intermediately resistant to AMC and recovered from urine (88.9%), blood (4.4%), wounds (4.4%) and ascitic fluid (2.2%), remained susceptible to first-generation cephalosporins and underwent further studies. PCR and nucleotide sequencing analysis revealed that 19 isolates produced the -lactamase IRT-11 (TEM-40) and the other 26 were OXA-1producers, whereas all harboured the bla SHV-1 gene. The percentage of AMC-nonsusceptible isolates dropped from 46.6% in 2006 to 14.3% in 2008.

A c c e p t e d M a n u s c r i p t

The 19 IRT-11-producing isolates were susceptible to TZP, extended-spectrum cephalosporins, carbapenems, gentamicin, tobramycin and amikacin, whilst resistance to other antibiotics assayed was variable, with fosfomycin (26.3%), ciprofloxacin (26.3%) and nalidixic acid (57.9%) showing the highest rates of resistance. All OXA-1-producing isolates were susceptible to cefotaxime, ceftazidime, cefepime (MIC between 1 mg/L and 4 mg/L in 61.5% of isolates), carbapenems and gentamicin and were resistant to tobramycin, kanamycin and ciprofloxacin.

Resistance rates to sulfonamides, nitrofurantoin, chloramphenicol, nalidixic acid and TZP were 76.9%, 80.8%, 84.6%, 92.3% and 92.3% and were <25% for the remaining antimicrobials studied.

Molecular epidemiology and detection of integrons, qnr genes and mutations in the QRDRs of gyrA and parC genes

The characteristics (ERIC-PCR pattern, resistance phenotype, presence and content of class 1 integrons, presence of qnr, amino acid changes in GyrA and ParC, and year of isolation) of the 19 IRT-11-and 26 OXA-1-producing isolates are shown in Table 2.

Three ERIC-PCR patterns were observed among the IRT-11-producing isolates, with E1 being the most prevalent (12; 63.2%), followed by E3 (5; 26.3%) and E2 (2; 10.5%). None of the IRT-11-producing isolates possessed qnr genes and only 1 presented a class 1 integron with a 2 kb/dfrA12-orf-aadA2 variable region.

OXA-1-producing isolates could be classified into three different groups. The first group comprised 20 isolates that carried a typical class 1 integron with the following isolates that shared the same ERIC-PCR pattern (E4) and harboured qnrS2 and a class 1 integron whose variable region was identical to that described above, although they were negative for qacE1 and sul1 (quaternary ammonium compounds and sulfonamide resistance genes associated with 3'-conserved region of class 1 integrons) as well as for sul3, a sulfonamide resistance gene encountered in the 3'-end of some atypical class 1 integrons [START_REF] Mo | Multidrug resistance related to class 1 integrons in human Salmonella enterica serotype Typhimurium isolates and emergence of atypical sul3-associated integrons[END_REF]. Finally, the third group was made up of another three sulfonamide-susceptible isolates that contained the aac(6')-Ib-crbla OXA-1 array but were negative for intI1, qnrA, qnrB and qnrS and were assigned to the E5 ERIC-PCR pattern. None of the OXA-1-producing isolates possessed orf513.

All nalidixic acid-resistant isolates analysed presented at least one point mutation in the gyrA or parC genes leading to amino acid substitutions.

Transfer of amoxicillin/clavulanic acid resistance and characteristics and antimicrobial resistance of transconjugants

Conjugation assays were carried out in selected IRT-11-and OXA-1-producing isolates displaying the different ERIC-PCR patterns identified in this work. Transfer by conjugation of genes involved in AMC resistance (bla IRT-11 or bla OXA-1 ) was achieved in all the isolates studied. Moreover, bla OXA-1 was systematically cotransferred with aac(6')-Ib-cr, and transconjugants from integron-carrying OXA-1producing isolates were positive for qnrS2 and bore class 1 integrons that were 3.

Discussion

The results of this work demonstrate that the high prevalence of decreased susceptibility to AMC among K. pneumoniae isolates recovered over a 3-year period from patients from a chronic care hospital was mediated by the production of IRT-11 or OXA-1, two penicillinases weakly inhibited by clavulanic acid, and was mainly due to the simultaneous spread of two different clones, one of which comprised isolates that produced IRT-11 (E1) and the other one comprising isolates that had acquired either the bla OXA-1 gene placed within a class 1 integron and linked to qnrS2 or the bla IRT-11 gene (E3).

In this series, isolates harbouring the bla IRT-11 gene located in a conjugative plasmid accounted for 37.3% of AMC-resistant K. pneumoniae isolates and for up to 11.1% of total isolates. IRT-11-producing isolates were distributed in three different clones, a finding which indicates that, in addition to clonal dissemination, horizontal transfer or independent emergence of bla IRT-11 may have occurred. Although IRT -lactamases have been described in sporadic clinical isolates of K. pneumoniae [START_REF] Lemozy | First characterization of inhibitor-resistant TEM (IRT) -lactamases in Klebsiella pneumoniae strains[END_REF], these enzymes, unlike TEM-derived ESBLs, are still infrequent and occur chiefly in community-acquired E. coli isolates [START_REF] Mo | Mechanisms of reduced susceptibility to amoxycillin-clavulanic acid in Escherichia coli strains from the health region of Tortosa (Catalonia, Spain)[END_REF][START_REF] Miró | Prevalence of clinical isolates of Escherichia coli producing inhibitor-resistant -lactamases at a university hospital in Barcelona, Spain, over a 3-year period[END_REF][START_REF] Martín | Population analysis and epidemiological features of inhibitor-resistant-TEM--lactamase-producing Escherichia coli isolates from both community and hospital settings in Madrid, Spain[END_REF]. The high diversity of mutant enzymes and the lack of clonal relatedness observed in some studies suggest the independent emergence and selection of these TEM variants under antibiotic selective pressure, posed by increasing use of inhibitor-containing combinations in clinical practice, rather than an epidemic dissemination [START_REF] Miró | Prevalence of clinical isolates of Escherichia coli producing inhibitor-resistant -lactamases at a university hospital in Barcelona, Spain, over a 3-year period[END_REF][START_REF] Martín | Population analysis and epidemiological features of inhibitor-resistant-TEM--lactamase-producing Escherichia coli isolates from both community and hospital settings in Madrid, Spain[END_REF]. Although IRT enzymes are usually A c c e p t e d M a n u s c r i p t encoded by genes harboured by conjugative plasmids, they appear to be less prone to be transmitted or selected than ESBLs or TEM-1 -lactamases, perhaps because their production constitutes a less efficient resistance mechanism, protecting bacteria specifically against the action of inhibitors but not against the effects of other lactams. In fact, to our knowledge there is only one previous description reporting an outbreak involving IRT -lactamases. This outbreak was caused by the dissemination of clonally related K. pneumoniae isolates recovered in a geriatric facility in France that produced IRT-2 [START_REF] Girlich | Molecular epidemiology of an outbreak due to IRT-2 -lactamase-producing strains of Klebsiella pneumoniae in a geriatric department[END_REF]. It is appropriate to point out that the isolates in the current study were also obtained from patients from a chronic care centre, a setting where bacteria are submitted to heavy antibiotic selection pressure and where strict adherence to infection control precautions may be difficult to achieve.

With regard to isolates bearing the bla OXA-1 gene, which represented 51% of AMCnon-susceptible isolates and 15.2% of total isolates, all of them also carried aac(6')-Ib-cr, a gene encoding a new variant of the acetyltransferase Acc(6')-Ib that exhibits the ability to acetylate not only tobramycin, kanamycin and amikacin but also quinolones with a piperazinyl amine such as ciprofloxacin. In most of these isolates the aac(6')-Ib-cr-bla OXA-1 cassette array was located within the variable region of a class 1 integron together with catB3 and arr3, which confer resistance to chloramphenicol and rifampicin, respectively, and was associated with a qnrS2 gene, probably in the same plasmid, since this determinant was consistently co-transferred by conjugation with the former integron. The aforementioned variable region was identical to that described in various enterobacterial clinical isolates from a different geographical origin [START_REF] Quiroga | Complex class 1 integrons with diverse variable regions, including aac(6')-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial isolates from Argentina[END_REF][START_REF] Chang | Characterization of class 1 integrons and gene cassettes in clinical isolates of Klebsiella pneumoniae from Taiwan[END_REF] and to that recently identified in the class 1 integron of an aquatic isolate of Aeromonas allosaccharophila [START_REF] Picão | Plasmid-mediated quinolone resistance in Aeromonas allosaccharophila recovered from a Swiss lake[END_REF]. However, when specified, the

A c c e p t e d M a n u s c r i p t

In37-like integron of clinical isolates was associated with qnrA or qnrB and with an ISCR1 element, whilst the integron of A. allosaccharophila was not linked to ISCR1 and was placed in a plasmid carrying qnrS2, similar to what occurred with the present K. pneumoniae isolates. Interestingly, three of the OXA-1-producing isolates obtained in 2008 carried a class 1 integron lacking both the qacE1 and sul1 genes typically present in the 3'-CS of class 1 integrons and that was also not associated with sul3. This circumstance also appears to be common among aquatic environmental bacteria, perhaps because natural bacterial isolates are less likely to have been exposed to synthetic antimicrobials such as sulfonamides and hence may not yet have found a need to integrate a sul resistance gene cassette into their integron [START_REF] Rosser | Identification and characterization of class 1 integrons in bacteria from an aquatic environment[END_REF]. Although genotyping analysis demonstrated that the current spread of OXA-1-producing K. pneumoniae isolates in the chronic care centre under discussion was the result of clonal dissemination, the fact that we have recently identified two qnrS2-positive E. coli isolates, likewise carrying aac(6')-Ib-cr, bla OXA-1 , catB3 and arr3 genes inserted in the variable region of a defective class 1 integron (unpublished data), in patients admitted to the former institution is of concern and evidences the role of integrons in the dissemination and co-selection of resistance in Gram-negative microorganisms.

Finally, we would like to highlight what in our opinion is the most striking finding of this work, namely the simultaneous emergence and scatter in a healthcare institution of K. pneumoniae isolates displaying two different resistance mechanisms, quite uncommon in this species, which confer specific resistance to BLIs. We speculate that the elevated consumption of AMC in the centre, where it was the most frequently prescribed antibiotic (data not shown), could have favoured the emergence and

(6')-B-R CTCGAATGCCTGGCGTGTTT sul1 Sul-F TGGTGACGGTGTTCGGCATTC Sul-R GCGAGGGTTTCCGAGAAGGTG sul3 Sul3-F CATTCTAGAAAACAGTCGTAGTTCG Sul3-R CATCTGCAGCTAACCTAGGGCTTTGGA qnrA QnrA-F ATTTCTCACGCCAGGATTTG QnrA-R GATCGGCAAAGGTTAGGTCA qnrB QnrB-F GATCGTGAAAGCCAGAAAGG QnrB-R ACGATGCCTGGTAGTTGTCC qnrS QnrS-F ACGACATTCGTCAACTGCAA QnrS-R TAAATTGGCACCCTGTAGGC qacE1 Qac-F GGCTGGCTTTTTCTTGTTATCG Qac-R TGAGCCCCATACCTACAAAGC orf513 orf513-F ATGGTTTCATGCGGGTT orf513-R CTGAGGGTGTGAGCGAG intI1 IntI1-F GGGTCAAGGATCTGGATTTCG IntI1-R ACATGGGTGTAAATCATCGTC

*

  Corresponding author. Tel.: +34 977 519 103. E-mail address: marolgap@terra.es (M.O. Pérez-Moreno).

A

  c c e p t e d M a n u s c r i p t variable region [3.6 kb/aac(6')-Ib-cr, bla OXA-1 , catB3, arr3] as well as qnrS2 and were ascribed to two closely related ERIC patterns (E3 and E3a) that differed from each other by only one band. The second group comprised three sulfonamide-susceptible

A

  c c e p t e d M a n u s c r i p t identical to those presented by donor strains. The MIC and other characteristics of the donor strains and their transconjugants are shown in Table

A

  c c e p t e d M a n u s c r i p t selection of IRT-11-and OXA-1-producing isolates. Additionally, in the latter case, the widespread use of fluoroquinolones could have also contributed, as is suggested by the association of bla OXA-1 with the plasmid-mediated quinolone resistance determinants aac(6')-Ib-cr and qnrS2 and the coexistence of chromosomal mutations in the QRDR. Moreover, deficiencies in infection control measures probably led to their propagation among patients, resulting in the consequent epidemic outbreak.

u s c r i p t 2 a

 2 Resistance phenotypes: Am, amoxicillin; Amc, amoxicillin/clavulanic acid; Nal, nalidixic acid; Fos, fosfomycin; C, chloramphenicol; Cip, ciprofloxacin; Nf, nitrofurantoin; K, kanamycin; Sxt, sulfamethoxazole/trimethoprim; T, tetracycline; St, streptomycin; Sp, spectinomycin; Sul, sulfonamides; Tob, tobramycin; Crx, cefuroxime; Ak, amikacin. Additional antibiotic resistances found in some but not all of the isolates are indicated by parentheses.b Size and resistance genes inserted within the variable region of class 1 integrons identified by PCR amplification and nucleotide sequencing using 5'-CS plus 3'-CS set of primers or 5'-CS plus bla OXA-1 reverse primers and 3'-CS or arr3 reverse primers plus bla OXA-1 forward primers:dfrA12 (trimethoprim resistance); aadA2 (streptomycin and spectinomycin resistance); aac(6')-Ib-cr (kanamycin, tobramycin and amikacin resistance and ciprofloxacin decreased susceptibility); bla OXA-1 (-lactam resistance); catB3 (chloramphenicol resistance), arr3 (rifampicin resistance).c Amino acid substitutions in GyrA and ParC deduced from mutations detected by PCR amplification and nucleotide sequencing of the quinolone-resistance determining regions of gyrA and parC genes from nalidixic acid-resistant isolates.d All isolates additionally produced SHV-1. e A single isolate. f Isolates with atypical 3'-end class 1 integron [negative for qacE1, sul1 and sul3 (quaternary ammonium compounds and sulfonamide resistance genes associated with class 1 integrons)].A c c e p t e d M a n u s c r i p t 1

Table 1

 1 Primers used in this study for polymerase chain reaction (PCR)

	Target	Primer	Sequence (5'3')
	bla TEM	TEM-1	ATA AAATTCTTGAAGACGAAA
		TEM-2	GACAGTTACCAATGCTTAATCA
	bla SHV	SHV-F	CACTCAAGGATGTATTGTG
		SHV-R	TTAGCGTTGCCAGTGCTCG
	bla OXA	OXA-F	AGCAGCGCCAGTGCATCA
		OXA-R	ATTCGACCCCAAGTTTCC
	bla PSE	OC-1	AATGGCAATCAGCGCTTC
		OC-2	GGGGCTTGATGCTCACTC
	aac(6')-Ib ACC(6')-B-F TTGCGATGCTCTATGAGTGGCTA
		ACC

Table 3

 3 Characteristics and resistance profiles of donor strains and transconjugants PCR, enterobacterial repetitive intergenic consensus polymerase chain reaction; MIC, minimum inhibitory concentration; Am, amoxicillin; Amc, amoxicillin/clavulanic acid; Tzp, piperacillin/tazobactam; Cfz, cefazolin; Cx, cefoxitin; Crx, cefuroxime; Ctx, cefotaxime; Cfp, cefepime; Tob, tobramycin; Ak, amikacin; Nal, nalidixic acid; Cip, ciprofloxacin; C, chloramphenicol; Nf, nitrofurantoin; Fos, fosfomycin; Sxt, sulfamethoxazole/trimethoprim.

	Isolate (-	IntI1/qnrS2	MIC (mg/L)
	lactamase;	a	Am Amc Tzp	Cfz Cx Crx Ctx	Cfp Tob Ak Nal Cip	C Nf	Fos Sxt
	ERIC-PCR
	pattern)
	EC J53 Az-R b No/No	≤4	≤4/2 ≤16/4 ≤2	≤4	≤1	≤0.12 ≤0.5 ≤2	≤4 ≤4	≤0.12 ≤4 ≤64 ≤16 ≤2/38
	Kp9 (IRT-11;	No/No	>16 16/8 ≤16/4 ≤2	≤4	≤1	≤0.12 ≤0.5 ≤2	≤4 ≤4	≤0.12 >8 ≤64 >32 ≤2/38
	E2) c
	TC Kp9 d	No/No	>16 16/8 ≤16/4 ≤2	≤4	≤1	≤0.12 ≤0.5 ≤2	≤4 ≤4	≤0.12 ≤4 ≤64 ≤16 ≤2/38
	Kp12 (IRT-11;	No/No	>16 16/8 ≤16/4 8	16	>16 ≤0.12 ≤0.5 ≤2	≤4 >16 >4	>8 >64 >32 ≤2/38
	E3) c
	TC Kp12 d	No/No	>16 16/8 ≤16/4 ≤2	≤4	≤1	≤0.12 ≤0.5 ≤2	≤4 ≤4	≤0.12 ≤4 ≤64 ≤16 ≤2/38
	Kp13 (IRT-11;	No/No	>16 16/8 ≤16/4 ≤2	≤4	≤1	≤0.12 ≤0.5 ≤2	≤4 ≤4	≤0.12 ≤4 ≤64 ≤16 ≤2/38
	E1) c
	TC Kp13 d	No/No	>16 16/8 ≤16/4 ≤2	≤4	≤1	≤0.12 ≤0.5 ≤2	≤4 ≤4	≤0.12 ≤4 ≤64 ≤16 ≤2/38
	Kp15 (IRT-11;	+/No	>16 16/8 ≤16/4 ≤2	≤4	2	≤0.12 ≤0.5 ≤2	≤4 >16 >4	>8 >64 16	>4/76
	E3) c
	TC Kp15 d	+/No	>16 16/8 ≤16/4 ≤2	≤4	≤1	≤0.12 ≤0.5 ≤2	≤4 ≤4	≤0.12 ≤4 ≤64 ≤16 >4/76

Acknowledgments

The authors thank M. Pilar Cid Ventura and Carmen López Escorihuela for excellent technical support in the conduct of this study as well as Donna Pringle for editorial assistance. They also thank J. Calvo Montes (Hospital Marqués de Valdecilla, Santander, Spain) for kindly providing E. coli J53-AzR.

Funding

This work was partially supported by Fundació Doctor Ferran (2006 Research on

Health Sciences Grant). The research of JR is supported by project CP05/0130 of the Fondo de Investigaciones Sanitarias (Spain).

Competing interests

None declared.

Ethical approval

Not required.


Ester B Aliyu

D Shitanda

S Walker

B Agnew

S Masheiti

R Atan

Performance and Exhaust Emissions of a Diesel Engine Fuelled with Croton Megalocarpus (Musine) Methyl

Keywords: Croton Megalocarpus, Musine, Bio-fuel, Methyl Ester, Engine test

The performance of a 4 stroke 3 cylinder direct injection naturally aspirated, Perkins D3.142 engine was measured in order to determine the suitability of a bio-fuel produced from the seeds of Croton Megalocarpus for engine use. The raw oil from the seeds was subjected to a transesterfication process and supplied to the engine as a croton methyl ester (CME) in various blends with diesel fuel. The engine performance (brake thermal efficiency, brake specific fuel consumption and exhaust temperature) were measured and evaluated. A Horiba PG-250 portable analyser was used to measure the CO, HC, CO 2 , NO x , and O 2 , concentrations. The tests indicated a lower brake thermal efficiency for CME compared with the pure diesel fuel. The exhaust gas temperature increased with increase in load for all tested fuels. It was found that the performance of the CME was comparable to pure diesel fuel but the bio-diesel produced lower smoke and NO x emissions. Emissions of CO were reduced at higher loads with the bio-fuel. The performance of the engine indicated that CME has properties and characteristics that make it a viable additive or alternative to conventional fossil diesel fuel.

Introduction:

As global oil reserves decline and climate change resulting from the burning of fossil fuels becomes more apparent, it has become necessary to develop and exploit sustainable and non-polluting (i.e. carbon neutral) energy sources. At the same time it has been estimated that oil production will show a downward trend to become just 35% of today's production by the year 2075. It is therefore necessary to find alternative sources of renewable energy if present energy consumption (and living standards) is to be maintained. Of the possible renewable sources bio-fuels are the only viable choice for use in transport systems that do not require huge investment in infrastructure and vehicle design. But some biomass alternatives such as ethanol, methanol and bio-diesel derived from food crops such as palm oil and rape seed oil are not without their critics. The consumption of biodiesel in the USA alone in 2006 was of the order of 250 million US gallons, most of which was produced from food crops or was grown on land previously used for food or animal feed production. In Europe pump diesel now contains 5% biofuel. In Indonesia large tracts of Equatorial forest are being destroyed to make way for palm oil production that is destroying the local ecosystem and it has been reported that the overall carbon signature of palm oil is actually greater than coal. There is now concern about moral and ethical issues associated with the substitution or use of food crops for bio fuel production [START_REF] David | Ethanol production using corn, switchgrass, and wood; biodiesel production using soybean and sunflower[END_REF]. The work reported in this paper examined a non food, environmentally friendly source of biodiesel, the Croton Megalocarpus Tree. Croton Megalocarpus is a tree from the family Eurhorbiaceae that grows generally in East Africa at an altitudinal range of 4,000 -6,000 ft. The tree grows up to 15 -35 m high [START_REF] Chudnoff | Tropical timbers of the world[END_REF], but can reach a height of 40 m or more [START_REF] Dale | Kenya trees and shrubs[END_REF], figure 1.

Figure 1 A Croton Megalocarpus tree grown as an ornamental plant in Kenya

The wood from the tree is of medium weight, hard, termite resistant and strong; it is used as a source of timber and fencing material. The leaves have high levels of nitrogen and phosphorous and serve as a source of mulch in plantations [START_REF] Beentje | Kenya trees, shrubs and lianas[END_REF]. Each fruit contains 3 ellipsoid ovoid or oblong-ellipsoid seeds which have an oil content of 30% by mass and a large proportion of protein. The Croton tree can survive on marginal agricultural land, not suitable for food production and can be planted to resist desert encroachment. The high protein content of the seed husks make them a very suitable poultry feed. The oil from the seeds can be extracted by mechanical extraction methods (pressing) or by solvent/chemical extraction methods. Presses such as manual screw, stork hydraulic hand press, motor jack press, mechanical press and a combination of these presses are available. Solvent extraction is done after preparatory steps such as crushing, blending and milling. The solvent is then added into the resulting paste and stirred until well mixed. The resulting solution is then filtered and the solvent driven off and recovered by a process of distillation to leave the raw vegetable oil. The seeds used in this study were obtained from the Kenya Forestry Research Institute Seed Centre in Kenya. The oil was obtained by both mechanical and chemical extraction methods in Kenya and then transported to Newcastle to be subjected to transesterification to be converted to biodiesel. The composition of the resulting fuel was determined by examination in a mass spectrometer and a sample was sent to the Fuel Oil Bunker Analysis Services (FOBAS) for analysis to determine the ignition and combustion characteristics in a constant volume bomb test according to IP541/06. Other properties determined at FOBAS included the density (EN ISO 3675) the sulphur content (EN ISO 20846r), the viscosity (EN ISO 3104) and the ash content (EN ISO 3451). The Croton methyl ester contains predominantly Oleic acid ester, with lesser quantities of Linoleic, Linolenic and Palmitic esthers. The properties of CME compared to commercially available red diesel (RD) fuel (EN590-Directive 98/70/CE) and a biodiesel derived from recycled vegetable oil (RV) [START_REF] Roskilly | The performance and the gaseous emissions of two small marine craft diesel engines fuelled with biodiesel[END_REF] are shown in table 1.

Table 1. Properties of CME compared with red diesel and biodiesel produced from recycled vegetable oil [START_REF] Roskilly | The performance and the gaseous emissions of two small marine craft diesel engines fuelled with biodiesel[END_REF].

Experimental Equipment and Tests:

The Engine used for the tests was a Perkins D3.152 engine, the main features of which are presented in the table 2 and displayed in figure 2. The air consumption of the engine was measured using the air box method and the cylinder pressure was sensed by a Kistler Piezo pressure transducer (type 401). The transducer was calibrated against a dead weight tester prior to the test. The engine was coupled to a hydraulic dynamometer through a BHL torque transducer. Load was applied to the engine by adjusting a control valve that set the delivery pressure of the dynamometer. The torque and speed of the engine were measured and from these the engine output power determined. Thermocouples (type K fast response) were used to take various temperature measurements on the test bed. The overall air fuel ratio was calculated by the Horiba analyser. Following the method adopted by Pramanik [START_REF] Pramanik | Properties and use of jatropha curcas oil and diesel fuel blends in compression ignition engine[END_REF] and Puhan etal [START_REF] Puhan | Mahua oil (Madhuca Indica seed oil) methyl ester as biodiesel-preparation and emission characterstics[END_REF] the test fuel was blended at different levels with conventional base diesel fuel. The mixtures contained 5 %, 10 %, 20 %, and 50 % CME (designated as B5, B10, B20 and B50) on a volume basis and tests were also performed with 100 % CME and 100 % red diesel (designated B100 and D100 respectively). Fuel flow measurements were made using an ISO-certified graduated cylinder connected to the fuel inlet piping via a three-way valve. A second three-way valve allowed fuel to be diverted from the fuel return line to the graduated cylinder thus forming a closed loop and ensuring that only the fuel consumed by the engine was measured. At each engine setting the time to consume a measured quantity of fuel was recorded. The exhaust flue gas concentrations of CO, HC, CO 2 , O 2 and NO x were measured with an Horiba PG-250 analyse which was calibrated at the start and the end of Table 2 Data of the Perkins Engine Figure 2 Test bed layout each days testing with span gases and the drift determined. The drift is a result of two factors, zero drift due to a change in ambient conditions and span drift produced by a change in the sensitivity of the instrument. The drift per hour was determined from the initial and final calibration and used to correct the experimental readings. The zero calibration was established using Nitrogen as a zero gas and the composition of the span gases is shown in table 3.

Table 3 Span gas concentrations

The systematic uncertainty of the analyser is ±0.5%, ±2% and ±1% in terms of repeatability, linearity and drift. The expected level of random uncertainties is shown in table 4. The smoke level was measured using a Bosch RTT100 smoke meter. Table 4 random uncertainties of gas emission measurements 3.0 Engine Test Procedure: The test performed was in conformity to ISO 8178-4, type E5 for diesel powered marine craft of less than 24 m in length. The test load points for this test are shown in table 5. The weighting factor is used to calculate specific exhaust emissions. The engine was warmed up at a light load setting with 100% diesel fuel until thermal stability was established in conformity with the manufacturer's recommendations. The test fuel was then delivered to the engine which was allowed to run for several minutes to clear the common parts of the fuel system of the pure diesel fuel. The load on the engine was then set at the test condition by adjusting the control valve on the dynamometer and at the same time the fuelling was adjusted to bring the engine to the test speed. The exhaust gas temperature; inlet water temperature; outlet water temperature and water jacket temperature were measured and monitored throughout the test. The fuel consumption was measured once the engine was stabilised at the operating point. Throughout the test the ambient air temperature and humidity readings were recorded to facilitate NO x correction in accordance to ISO 8178-1 to normalise the test readings to standard ISO conditions with humidity of 10.71 g/kg and ambient temperature of 25 o C. The brake thermal efficiency of the engine with diesel fuel and the various blends of CME with red diesel (B5, B20, B50 and B100) was determined from the measured results and plotted against brake power output as shown in figure 3. The test results show that with increasing brake power the thermal efficiencies of all the fuels show similar trends with the straight diesel fuel showing the better performance over the whole power/speed range. The 100 % CME showed the worst performance. The drop in thermal efficiency with increase in the blend of CME-bio-diesel can be attributed to the higher fuel supply for a given power which produces slower combustion times and counteracts the possible benefit of the presence of fuel borne oxygen in enhancing the combustion process. The combustion chamber and injection characteristics of the test engine were optimised for operation with red diesel so it is possible that small modifications to the injection timing may improve the situation for the bio-fuel. The calorific value of CME oil is 40.280 MJ/kg as compared to the 42 MJ/kg for the diesel fuel. These results are in general agreement with those of Pramanik [START_REF] Pramanik | Properties and use of jatropha curcas oil and diesel fuel blends in compression ignition engine[END_REF] and Puhan et al [START_REF] Puhan | Mahua oil (Madhuca Indica seed oil) methyl ester as biodiesel-preparation and emission characterstics[END_REF][START_REF] Puhan | Performance and emission study of Mahua oil (madhuca indica oil) ethyl ester in a 4-stroke natural aspirated direct injection diesel engine[END_REF] for other bio-fuel diesel blends.

Figure 3 Brake thermal efficiency (%) vs. engine power for the test fuels

The brake specific fuel consumption (bsfc) of the CME varies depending on the engine power and speed. For all the fuels tested the consumption was high at low speed but decreased with increasing speed. This is shown in figure 4. At high speeds, friction heat losses and deteriorating combustion increases bsfc.

Although the blend gave almost the same power as red diesel, its bsfc is higher. This can be attributed to the oxygen content of the CME which takes no part in the combustion process and reduces the heating value compared with red diesel fuel. With an increase in biodiesel percentage in the blends, the calorific value of the mixture decreases. Hence the specific fuel consumption of the higher percentage of biodiesel in blends also increases. It can be concluded that the amount of fuel required to produce the same amount of power output varies from oil type and the loading conditions. Raheman and Gadge [START_REF] Raheman | Performance of compression ignition engine with mahua (Madhuca indica) biodiesel[END_REF] reported that higher densities resulted in higher values for bsfc. As the density of B100 was higher than that of diesel fuel, it means the same fuel consumption on a volume basis resulted in higher bsfc as the proportion of CME is increased. The variation of exhaust temperature with bmep is shown in figure 5. The highest exhaust gas temperature of 448 o C was recorded for the B100 at the maximum power of 30kW. The least exhaust temperature of 165 o C was with D100 at 7.5 kW power rating. There is a steady rise of temperature as the power and speed are increased on the engine. The B5, B20 and B50 have almost a similar rating of exhaust heat. The poorer combustion characteristic of the CME as a result of its higher viscosity compared to the red diesel was assumed to be the cause. The higher exhaust temperature of the Croton SME is indicative of lower thermal efficiency, less of the energy input in the fuel was converted to useful work, and the remaining heat is given out as exhaust temperature [START_REF] Pramanik | Properties and use of jatropha curcas oil and diesel fuel blends in compression ignition engine[END_REF]. Puhan [START_REF] Puhan | Performance and emission study of Mahua oil (madhuca indica oil) ethyl ester in a 4-stroke natural aspirated direct injection diesel engine[END_REF] considered that the high exhaust temperature results from longer ignition delays that produce a higher exhaust temperature but Al-Widyan et al, [START_REF] Al-Widyan | Utilization of ethyl ester of waste vegetable oils as fuel in diesel engines[END_REF] stated that the higher exhaust temperatures may result from higher combustion temperatures. The emissions of Carbon Monoxide (CO) from the test engine when fuelled with the diesel and the CME and its blends are compared in figure 6. The CO emissions of the blends were higher than those of the Diesel fuel at the higher load and speed settings but were lowest at the lower speed and power settings. The increase of the CME percentage in the fuel blend reduces the CO at the low load setting as the B100 has the least CO but the reverse is the case with the higher load settings. There are several variables acting on the combustion process that has an impact on CO formation. The additional Oxygen present in the CME will assists in making the combustion more complete in the combustion zone but at the high load condition the increased viscosity of the CME could affect the fuel injection and atomisation characteristics could adversely impact on the CME as the injector was optimised for Diesel fuel. The increased in-cylinder temperatures at high load would have a greater impact on the CO production of Diesel fuel due to the enhanced atomisation and mixing this produces. Modifications to the fuel injection system may improve the atomisation characteristics of the CME and hence improve the combustion at high load.

Figure 6 Variation of CO emissions with speed and load for CME and its blend with red diesel Figure 7 shows the differences in HC emission between the blends of the biodiesel and the red diesel. The recorded emission levels for the biofuel are comparatively low and reduce with increased engine speed until at 2200 rpm they increased. This has been observed for other biofuels [START_REF] Al-Widyan | Utilization of ethyl ester of waste vegetable oils as fuel in diesel engines[END_REF] for reasons related to the oxygen content of the fuel. Studies by Monyem etal [START_REF] Monyem | The effect of biodiesel oxidation on engine performance and emissions[END_REF] show that unburned hydrocarbons generally emanate from the regions in a cylinder where there is a poor fuel/air mixture to such a high extent that the ratio is past the combustion limit. This process would be less likely to occur with fuel borne oxygen that can extend the combustion limit and reduce the extent of fuel quench. The B20 has shown a consistent low level of HC emission in this study compared to both the B100 and D100 at 1380 rpm and 1760 rpm but slightly higher than the diesel at 2002 and 2200 rpm. Without information about the complex in-cylinder combustion chemistry further considerations on this matter are just speculation but the results are encouraging.

Figure 7 Variation of HC emissions with speed for CME and its blends with red diesel

There was a general increase of NOx emissions as load and speed were increased. This increase in NOx is consistent with increase in cylinder temperature and exhaust temperature but the emissions for the bio-fuel were lower than the red diesel fuel at higher operating loads as shown in figure 8. These results are in agreement of those found by Roskilly et al [START_REF] Roskilly | The performance and the gaseous emissions of two small marine craft diesel engines fuelled with biodiesel[END_REF]. At low loads the overall oxygen concentration is higher but the combustion temperature is lower and (a feature of the chosen test method) the combustion time is longer which could both lead to low NOx but the fuel borne oxygen is most effective at producing NOx in the reaction zone which aided by the high local temperatures will then have more time to revert back to an equilibrium concentration [START_REF] Usta | Combustion of biodiesel fuel produced from hazelnut soapstock/waste sunflower oil mixture in a Diesel engine[END_REF].

Figure 8 Variation of NOx emissions with speed and load for CME and its blends with red diesel. It has been observed from the result that at low load and speed the smoke emission is best with the red diesel fuel as a result of perhaps more favourable viscosity and density of this fuel as shown in figure 9. As the engine load and speed is increased, the smoke produced by the CME is initially higher than the red diesel for all blends but as the load is increased the bio-fuel begins to have a reduced level of emissions compared with the red diesel. The smoke emission of the Perkins engines for B100 is 20% less than that of red diesel, at high speed and load, but at the low speed and load that of the red diesel is the lowest. As more load was added, the temperature of the engine rises and makes combustion better for the CME. This is in agreement with the results of Mittlebach et al [START_REF] Mittelbach | Biodiesel: The comprehensive handbook[END_REF] who noticed that smoke density for biodiesel blend was generally lower than that of the diesel oil and they reasoned that higher thermal efficiency indicates better and complete combustion of fuel. Biodiesel has greater oxygen content than red diesel which may assist in reducing the smoke by locally increasing the constituent of oxygen available for combustion. Puhan et al [START_REF] Puhan | Performance and emission study of Mahua oil (madhuca indica oil) ethyl ester in a 4-stroke natural aspirated direct injection diesel engine[END_REF] reported a reduction of 70% smoke in case of Mahua oil methyl ester when compared to diesel. The fuel injection equipment and timing of the test engine were at the normal setting for red diesel fuel; it may be that the smoke level for the CME could be lowered at low load if the injection timing was optimised for this fuel. Carbon dioxide, (CO 2 ) has a great effect on the green house gases, in that it has the tendency to form a blanket effect on the atmosphere which aids global warming. The CO 2 generated with the CME at the various loads and speed is shown in figure 10. There is a steady increase of CO 2 with engine speed and load due to increased fuelling. The CO 2 from Croton is seen to be higher than that of diesel fuel (D100). This is consistent with the lower hydrogen/carbon ratio and lower calorific value of the bio fuel. The CO 2 emissions of the bio-fuel can however be considered as zero carbon emissions as they are sourced by the plant from air bourn carbon. The oxygen concentrations in the exhaust are shown in figure 11. The biofuel contains oxygen as part of its chemical make up which is not the case for red diesel fuel. It is expected that the oxygen content in the exhaust decreases as the engine load is increased as the air fuel ratio decreases with load. This paper has demonstrated that biodiesel from Croton Megalocarpus has a very strong potential for use as a biofuel for diesel engines in terms of power output, economy and exhaust emissions. The performance of a Perkins engine when fuelled with biofuel and biofuel/diesel blends is very similar to that when fuelled with red diesel. The main conclusions derived from this research are;

• The Croton biodiesel when blended with diesel fuel produces a level of performance in economy and exhaust emissions that are very similar to other biofuels from other sources. • The brake thermal efficiency of the engine when fuelled with pure CME and the blended fuels is lower than that of red diesel fuel for the particular engine tested. • The fuel consumption of B5, B20, B50 and B100 at 1380 rpm and 7.5 kW power was 404.03, 415.5, 420, 424 g/kWhr respectively. This represented an increase of 2.65%, 3.8%, 4.7% and 4.7% respectively over that of basic red diesel fuel. • CO, CO 2 and HC emissions were higher for the CME and blends but the smoke emission were lower when compared to the red diesel fuel.

Figure 4 .

 4 Figure 4. Brake specific fuel consumption vs. engine speed (rpm) for the test fuels.

Figure 5

 5 Figure 5 Exhaust Temperature vs bmep for the test fuels.

Figure 9

 9 Figure 9 Variation of Smoke emissions with speed and load for CME and its blends with red diesel.

Figure 10

 10 Figure 10 Variation of CO 2 emissions with speed and load for CME and its blends with red diesel

Figure 11

 11 Figure 11 Variation of exhaust O 2 content with speed and load for CME and its blends with red diesel

Figure 1 AFigure 2 Figure 3 Figure 4

 1234 Figure 1 A Croton Megalocarpus tree grown as an ornamental plant in Kenya

Figure 5 ExhaustFigure 6 Figure 7 Figure 8 Figure 9

 56789 Figure 5 Exhaust Temperature vs bmep for the test fuels

Figure 10 Figure 11

 1011 Figure 10 Variation of CO 2 emissions with speed and load for CME and its blends with red diesel

Figure 2 Figure 11

 211 Figure 2 Test bed layout.

Table 5

 5 Test mode cycle ISO 8178-4 type 5 4.0 Test Results and Discussions:

•

  The experimental results tentatively support the assertion that methyl esters of Croton Megalocarpus can be successfully used in existing diesel engines without modifications but this needs to be verified by further long term and comprehensive tests. Use of the bio-diesel as a partial fuel substitute can stimulate the rural farm economy in Africa, bring unproductive land back into profitable production, slow desert encroachment and make farming communities self reliant.

		CO (ppm)	CO 2 (%)	NO x (ppm) O 2 (%)
	Initial span	1385	13.91	1388	21.2
	Maximum span drift	63	0.34	87	0
	Maximum zero drift	1	0	0	0.16
	Full scale of analyser	2000	20	2500	100
	Span drift on full scale (%)	3.15	1.7	3.48	0
	Zero drift on full scale (%)	0.05	0	0	0.16

Table 5

 5

	Mode number (Cycle E5)	Mode 1	Mode 2	Mode 3	Mode 4	Mode 5
	Speed % of Max	100	91	80	63	Idle
	Power % of Max	100	75	50	25	0
	Engine speed rpm	2200	2002	1760	1380	500
	Engine power kW	33.56	25.17	16.78	8.39	-
	Engine Torque Nm	145	119	90.9	58.8	-
	Weighting Factor	0.08	0.13	0.17	0.32	0.3