Maria Lluch-Senar

Enrique Querol

Jaume Piñol
email: jaume.pinyol@uab.cat

Cell division in a minimal bacterium in the absence of ftsZ

Keywords: Mycoplasma genitalium, cell division, ftsZ, gliding motility Mycoplasma genitalium, ftsZ, cell division, gliding motility

Mycoplasma genomes exhibit an impressively low amount of genes involved in cell division and some species even lack the ftsZ gene, which is found widespread in the microbial world and is considered essential for cell division by binary fission. We constructed a Mycoplasma genitalium ftsZ null mutant by gene replacement to investigate the role of this gene and the presence of alternative cell division mechanisms in this minimal bacterium. Our results demonstrate that ftsZ is non-essential for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery. This is an alternative mechanism, completely independent of the FtsZ protein, to perform cell division by binary fission in a microorganism. We also propose that the mycoplasma cytoskeleton, a complex network of proteins involved in many aspects of the biology of these microorganisms, may have taken over the function of many genes involved in cell division, allowing their loss in the regressive evolution of the streamlined mycoplasma genomes.

INTRODUCTION

Mycoplasmas are characterized by having small genomes with a low G+C content and by the absence of a cell wall, which confers on them a pleomorphic appearance. These microorganisms, belonging to the Mollicutes class, are widespread in nature and they are parasites or pathogens of a very broad range of hosts, including humans. Although they have a minute and deceptively simple appearance, a closer study of mycoplasma cells reveals considerable intricacy. Despite their reduced genomes, many mycoplasmas have advanced systems required for a parasitic life such as adhesion and internalization inside host cells, antigenic variation, extensive membrane transport systems, or the ability to locomote over solid surfaces (gliding motility).

Mycoplasma genitalium is considered the etiological agent of non-gonococcal and nonchlamydial urethritis [START_REF] Jensen | Mycoplasma genitalium: the aetiological agent of urethritis and other sexually transmitted diseases[END_REF]. The most relevant structure in the M. genitalium cytoplasm is a cytoskeleton organized around a rod shaped electrondense core [START_REF] Hatchel | Attachment organelle ultrastructure correlates with phylogeny, not gliding motility properties, in Mycoplasma pneumoniae relatives[END_REF][START_REF] Pich | Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence[END_REF] that defines a distinctive tip structure known as terminal organelle. The majority of cytoskeletal proteins present in the terminal organelle have been primarily identified as cytadhesins or cytadherence-associated proteins [START_REF] Burgos | Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development[END_REF][START_REF] Burgos | Functional analysis of the Mycoplasma genitalium MG312 protein reveals a specific requirement of the MG312 N-terminal domain for gliding motility[END_REF][START_REF] Pich | Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence[END_REF]. However, a variety of proteins involved in energy metabolism (dihydrofolate reductase, DHFR), translation/transcription (Elongation factor Tu), heat shock (DnaK), and cell division (FtsZ) were also identified as cytoskeletal proteins in the closely related species Mycoplasma pneumoniae [START_REF] Regula | Defining the mycoplasma 'cytoskeleton': the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry[END_REF]. The terminal organelle is essential for cytadherence and further parasitism of host target cells [START_REF] Burgos | Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development[END_REF][START_REF] Mernaugh | Properties of adhering and nonadhering populations of Mycoplasma genitalium[END_REF] and has a key role in the guidance of the mycoplasma movement [START_REF] Burgos | Deletion of the Mycoplasma genitalium MG_217 gene modifies cell gliding behaviour by altering terminal organelle curvature[END_REF]. Compelling evidence also support that the terminal organelle is (or F o r P e e r R e v i e w contains) the molecular motor for gliding motility in M. pneumoniae [START_REF] Hasselbring | Proteins P24 and P41 function in the regulation of terminal-organelle development and gliding motility in Mycoplasma pneumoniae[END_REF]. Untreated M. genitalium infections can persist for a long time [START_REF] Cohen | Mycoplasma genitalium infection and persistence in a cohort of female sex workers in Nairobi, Kenya[END_REF][START_REF] Hjorth | Sequence-based typing of Mycoplasma genitalium reveals sexual transmission[END_REF]. Among the mechanisms allowing for such persistence, there are highly sophisticated systems to evade the host immune response. The most obvious one is probably the intracellular residence of this microorganism, but mechanisms to generate antigenic variation can also be found. It is well known that variability occurs in the P140 and P110 main adhesins, which are essential for the terminal organelle development [START_REF] Burgos | Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development[END_REF]. These adhesins are encoded, respectively, by ORFs mg191 and mg192 (also referred to as mgpB and mgpC), located inside the MgPa operon. Several non-coding sequences reminiscent of mg191 and mg192 also exist, scattered along the M. genitalium genome, and they are known as MgPa repeats or MgPa islands [START_REF] Fraser | The minimal gene complement of Mycoplasma genitalium[END_REF][START_REF] Peterson | Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation[END_REF]. Prominent in a densely packed genome of only 580 kb, these repetitive sequences provide a nearly unlimited source of antigenic variation by recombining amongst themselves and with the coding sequences in the MgPa operon [START_REF] Iverson-Cabral | mgpB and mgpC sequence diversity in Mycoplasma genitalium is generated by segmental reciprocal recombination with repetitive chromosomal sequences[END_REF][START_REF] Ma | Mycoplasma genitalium: an efficient strategy to generate genetic variation from a minimal genome[END_REF]. MgPa islands also endow a phase variation mechanism which can switch ON-OFF, in a reversible or irreversible way, the expression of P110 and P140 adhesins [START_REF] Burgos | Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development[END_REF]. These phase variants are also generated by recombination and they lose the terminal organelle, in consequence being unable to adhere to host cells or other surfaces.

It is currently thought that mycoplasmas evolved from bacteria of the Firmicutes taxon by regressive evolution [START_REF] Weisburg | A phylogenetic analysis of the mycoplasmas: basis for their classification[END_REF]. A parasitic way of life in very constant environments likely led ancient mycoplasmas to lose many of their genes, in this way modeling the minimal genomes that these microorganisms exhibit. One of the clearest examples of such reductive evolution can be seen when examining the operons or clusters containing genes involved in cell division. While it is very common to find 15 or 16 genes in the division and cell wall synthesis cluster (DCW) of cell-walled bacteria, most sequenced mycoplasma genomes include only mraZ, mraW, ftsZ and one gene encoding a hypothetical protein. It is also noteworthy that some mycoplasma species may even lack several of these genes. Despite the fact that a few other genes may also be collaborating in cell division [START_REF] Alarcón | Genes involved in cell division in mycoplasmas[END_REF], genes in the DCW of mycoplasmas are expected to be essential for cell growth. This view was reinforced when no disruptions were identified in the corresponding operons of M. genitalium, M. pneumoniae and M. pulmonis by global transposon mutagenesis [START_REF] French | Large-Scale Transposon Mutagenesis of Mycoplasma pulmonis[END_REF][START_REF] Glass | Essential genes of a minimal bacterium[END_REF][START_REF] Hutchison | Global transposon mutagenesis and a minimal Mycoplasma genome[END_REF]. However, in a previous work we found a transposon insertion inside the coding region of M. genitalium ftsZ [START_REF] Lluch-Senar | A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium[END_REF]. This gene was shown to be transcribed in M. genitalium [START_REF] Benders | Transcriptional analysis of the conserved ftsZ gene cluster in Mycoplasma genitalium and Mycoplasma pneumoniae[END_REF] and is considered essential for cell division by binary fission. The encoded tubulin-like FtsZ protein is the first component of the cell division apparatus that arrives at the division site and polymerizes at midcell, forming a filamentous ring (Z-ring) that recruits additional proteins required for the progression and the completion of cytokinesis [START_REF] Weiss | Bacterial cell division and the septal ring[END_REF].

The transposon insertion previously identified in ftsZ was compatible with the presence of a truncated protein containing a significant amount (40%) of the amino acid sequence. Thus, we aimed to construct a M. genitalium ftsZ null mutant by gene replacement to better understand the role of this protein. The obtained mutant strain was characterized in terms of growth kinetics, ultrastructure, cell adhesion and gliding motility. The results confirm our previous expectations indicating a non-essential role of ftsZ for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can still manage a feasible cell division and cytokinesis using the force generated by the gliding motility apparatus. To our knowledge, this is the first report depicting alternative mechanisms to perform cell division by binary fission in a microorganism that already contains the ftsZ gene.

RESULTS

Obtaining a M. genitalium ftsZ null mutant

M. genitalium ftsZ (ORF mg224) was deleted by gene replacement. The p∆ftsZTet suicide plasmid was designed to contain the tetM438 selectable marker enclosed by the flanking regions of the mg224 ORF (Fig. 1A). After transforming M. genitalium wildtype (WT) G37 strain with plasmid p∆ftsZTet, recombinant clones were analyzed by Southern blot (Fig. 1B). Two of ten clones showed the intended replacement as a result of a double-crossover recombination event between p∆ftsZTet and the mycoplasma genome, resulting in the complete deletion of the mg224 ORF (Fig. 1A). The absence of ftsZ in the null mutants, which were termed ∆ftsZ, was also confirmed by PCR and Southern blot (Fig. 1C,E). Importantly, plasmid p∆ftsZTet was designed to preserve promoter signals surrounding the mg224 ORF (Fig. 1A), in particular the previously identified promoter sequence upstream of mg225 ORF [START_REF] Benders | Transcriptional analysis of the conserved ftsZ gene cluster in Mycoplasma genitalium and Mycoplasma pneumoniae[END_REF]. To confirm that the deletion in ftsZ was not affecting the transcription of downstream genes, cDNA from mg225 ORF in ∆ftsZ mutants was consistently amplified by RT-PCR (Fig. 1D). The growth kinetics of the ∆ftsZ mutant was compared to that of the WT strain. The cell mass from several cultures of both strains was measured by ATP luminometry [START_REF] Lluch-Senar | A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium[END_REF][START_REF] Stemke | Measurement of Mollicute Growth bt ATP-Dependent Luminometry[END_REF]. Cultures of both strains exhibited the same doubling time in log phase (11.8 h and 11.7 h, respectively) and no significant differences were found when analyzing additional parameters such as the cell mass in the stationary phase. These results indicate that cells not only remain viable upon deletion of ftsZ, but also that this deletion has no impact on growth kinetics of M. genitalium.

Growth kinetics of ∆ftsZ cells

Adhesion properties and cytoskeletal structure of ∆ftsZ cells

Since FtsZ protein has previously been identified as a cytoskeletal protein in the related species M. pneumoniae [START_REF] Regula | Defining the mycoplasma 'cytoskeleton': the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry[END_REF], we were prompted to investigate the presence of defects in the adherence properties as well as downstream events affecting other cytoskeletal proteins in ∆ftsZ mutants. Haemadsorption (HA) was qualitatively assessed, and colonies from ∆ftsZ cells exhibited the same HA + phenotype as colonies from WT cells. ∆ftsZ cells did not show significant differences regarding their plastic adhesion properties (Table 1). Neither were there significant differences found when examining the protein profiles of ∆ftsZ and WT cells by SDS-PAGE and Western blot.

In particular, ∆ftsZ cells exhibited WT levels of MG386, HMW1, HMW2, P140, P110, HMW3 and P41 proteins (data not shown), indicating that deletion of ftsZ does not give rise to detectable downstream events in most of the cytoskeletal proteins of M. genitalium. In addition, examination of ultrathin sections of ∆ftsZ cells by transmission electron microscopy did not reveal appreciable ultrastructural changes in the electrondense core or other cytoskeletal components of the terminal organelle.

However, long cell extensions were frequently observed in the pole opposite to the terminal organelle of ∆ftsZ cells (Fig. 3A).

Gliding motility properties of ∆ftsZ cells

Because no significant changes were observed in the cytoskeleton of the mutant cells, no changes were expected in the gliding motility properties of ∆ftsZ cells. However, gliding motility was assessed by examining colony morphology in culture dishes covered with semisolid medium. Under these conditions, colonies from cells with gliding motility defects are compact and do not exhibit the microsatellite colonies that are typically developed from gliding proficient cells (Pich et al., 2006a). Surprisingly, colonies from ∆ftsZ cells were more compact and with fewer satellite microcolonies than those from WT cells (Fig. 3B), suggesting that deletion of ftsZ interferes in some way with the gliding motion. Gliding motility was also monitored by microcinematography. The speed and movement patterns of ∆ftsZ cells were essentially the same shown by WT cells, although it was noticeable that 39% of ∆ftsZ cells vs. 10% of WT cells remained non-motile during the observation period (Table 1). Interestingly, in the microcinematographies most non-motile ∆ftsZ cells were found very close to another non-motile cell, and sometimes both cells seemed to be linked by a thin filament (Fig. 3C). Thus, the high ratio of non-motile ∆ftsZ cells is consistent with the more compact phenotype of the colonies of this strain. However, the normal velocity and movement patterns exhibited by motile ∆ftsZ cells suggest that the FtsZ protein is not directly involved in the gliding mechanics of mycoplasma.

Analysis of dividing ∆ftsZ cells

To investigate the origin of non-motile cells detected in the ∆ftsZ strain, the mutant strain was analyzed by scanning electron microscopy (SEM). The images confirmed the presence of a large number of coupled cells linked by a thin filament (Fig. 4A andB) that were also observed in pictures from the cinematographies. Noteworthy, these coupled cells exhibited terminal organelles at both ends. Since the terminal organelle is the leading end in the mycoplasma motion [START_REF] Bredt | Motility and multiplication of Mycoplasma pneumoniae. A phase contrast study[END_REF][START_REF] Burgos | Deletion of the Mycoplasma genitalium MG_217 gene modifies cell gliding behaviour by altering terminal organelle curvature[END_REF], cells bearing two terminal organelles in the same direction but opposite senses are expected to be non-motile or barely motile. The images also suggest that these cells are dividing cells in the latter stages of cytokinesis, as has recently been demonstrated in M. pneumoniae (Hasselbring et al., 2006a). In addition, cells exhibiting a long tail in the pole opposite of the terminal organelle as well as chains of cells were also very common (Fig. 4A andB). Such morphologies were rarely observed when examining WT cells and are in agreement with the possibility that the force generated by the gliding motility apparatus in one or both daughter cells may be strong enough to separate dividing ∆ftsZ cells.

Introducing non-adherent mutations in the ∆ ∆ ∆ ∆ftsZ strain

If the gliding apparatus is essential for cytokinesis in ∆ftsZ cells, additional mutations abolishing motility in this strain should render cells unable to divide, thus being, in consequence, non-viable. Unfortunately, methods to construct strains bearing conditional lethal mutations are not currently available in mycoplasmas. However, we have taken advantage of the phase variation mechanism that deletes a region involving coding sequences of P110 and P140 adhesins [START_REF] Burgos | Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development[END_REF] to investigate the role of the gliding machinery in the cytokinesis of ∆ftsZ cells. Since P140 and P110

proteins are required for cell adhesion, phase variants are also expected to be nonmotile. Supporting this view, M. pneumoniae cells are non-motile after disruption of the gene coding for the P1 adhesin (Hasselbring et al., 2006b), homologous to the P140 adhesin. In addition, phase variants occur with high frequency in the WT strain and can Based on this rationale, the presence of HA -variants in serial culture passages of WT and ∆ftsZ strains was screened. Viable, non-adherent cells were detected when they were able to develop HA -colonies on agar plates [START_REF] Mernaugh | Properties of adhering and nonadhering populations of Mycoplasma genitalium[END_REF]. The frequency of phase variants was low at passage 0 of WT cultures (0.3%) but it increased rapidly until becoming stabilized with a frequency of around 2.8% in the last passages (Fig. 5A andB). In contrast, phase variants were never detected in the successive culture passages of the ∆ftsZ strain. This result indicates that HA -phase variants are not viable in the genetic background of the ∆ftsZ strain. The presence of the recombinant chromosomes from phase variants in the serial culture passages was also investigated.

For this purpose, a quantitative real time PCR (Q-PCR) was designed to detect a specific amplicon resulting from the recombination of the MgPa operon with MgPa island VI (Fig. 5C andD). Detection of this amplicon only reveals the presence of chromosomes from a fraction of non-adherent cells since additional recombination events rendering non-adherent cells are also possible with other MgPa islands [START_REF] Burgos | Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development[END_REF]. The identity of this amplicon was confirmed by sequencing (data not shown). At passage 0, the frequency of recombinant chromosomes was very similar in the cultures from both strains (0.1%). This result indicates that recombination events deleting a portion of the MgPa operon also occur in the ∆ftsZ strain and these events have a similar frequency in the WT strain. In WT cultures, the frequency of chromosomes with a deletion in the MgPa operon was progressively increased until reaching values close to 0.4% in the fourth passage and then remained stabilized until the end of the experiment (Fig. 5C). Cultures from the ∆ftsZ strain did not show such

Complementation analysis of the ∆ ∆ ∆ ∆ftsZ strain

Because only two ∆ftsZ mutants were isolated by gene replacement, a complementation assay of the ∆ftsZ strain was performed to exclude the presence of additional genetic defects in this strain. The WT ftsZ allele under the control of the mg438 constitutive promoter [START_REF] Burgos | Functional analysis of the Mycoplasma genitalium MG312 protein reveals a specific requirement of the MG312 N-terminal domain for gliding motility[END_REF] was reintroduced by transposition to ∆ftsZ cells by using plasmid pTnGftsZ (Table S2). After transforming with this plasmid, 58% of the recovered colonies exhibited normal growth, with the presence of a large number of microsatellites (Fig S1), suggesting that the WT ftsZ allele in ∆ftsZ cells was able to restore the normal gliding phenotype. Four colonies were picked, filtered-cloned and the corresponding cultures were analyzed by Southern blot to exclude the presence of multiple transposon insertions (data not shown). Microcinematographies from cells of two of these colonies confirmed that gliding motility parameters were restored to normal levels (Fig. S1). In the same way, SEM analyses demonstrated that frequencies of different morphologies were restored to values very close to those shown by WT cells (Fig. 4A). Finally, one of these transformants was submitted to successive passages and HA -phase variants were recovered again, with a very similar frequency to that observed in the WT strain (Fig. S1). Together, these data indicate that the presence of the WT ftsZ allele is able to restore the normal growth parameters in ∆ftsZ cells, and exclude that genetic defects other than the absence of ftsZ are involved in the phenotype exhibited by the ∆ftsZ strain. genitalium in depth and thus gain insight into its biology and cell division systems. The data reported herein demonstrate that M. genitalium cells can divide by binary fission in the absence of FtsZ and highlight the role of the terminal organelle and its associated cytoskeletal structures in the cell division of this minimal microorganism. This finding is comparable to the L-forms of Bacillus subtilis dividing by a "budding" or "extrusionresolution" mechanism in the absence of the ftsZ gene, probably reflecting the presence of ancient proliferative mechanisms in the modern microorganisms [START_REF] Leaver | Life without a wall or division machine in Bacillus subtilis[END_REF]. Our results suggest that in M. genitalium binary fission may be accomplished by two redundant mechanisms, the first one based on the well-known ftsZ machinery and the second one based on the pivotal roles of the mycoplasma cytoskeleton and gliding motility.

FtsZ has previously been identified as a component of the cytoskeletal fraction of M.

pneumoniae cells [START_REF] Regula | Defining the mycoplasma 'cytoskeleton': the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry[END_REF], in accordance with the biochemical properties of eukaryotic cytoskeletal tubulins [START_REF] Ramsby | Differential detergent fractionation of eukaryotic cells. Analysis by two-dimensional gel electrophoresis. 2-D Proteome Analysis Protocols[END_REF]. Nonetheless, cells from the ∆ftsZ strain do not exhibit adherence defects or detectable changes in their cytoskeletal ultrastructure and glide at the same velocity as do WT cells. Furthermore, ∆ftsZ cells do not show downstream events in their levels of proteins involved in cell adhesion and motility. These functional impairments and such downstream events are commonly observed in strains with deletions or disruptions in genes coding for functional cytoskeletal proteins in M. genitalium [START_REF] Burgos | Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development[END_REF][START_REF] Burgos | Deletion of the Mycoplasma genitalium MG_217 gene modifies cell gliding behaviour by altering terminal organelle curvature[END_REF][START_REF] Pich | Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence[END_REF] and M. pneumonaie [START_REF] Krause | Structure, function, and assembly of the terminal organelle of Mycoplasma pneumoniae[END_REF]. While our results do not discard the possible association of FtsZ with the main structures of the mycoplasma cytoskeleton, they exclude a role for this protein in the functions traditionally linked to the terminal organelle and its associated cytoskeletal structures.

SEM analysis of the ∆ftsZ strain reveals a high frequency of mutant cells in the latter stages of cytokinesis, suggesting that this process takes a long time in this strain (approximately four hours). In contrast, the low frequency of dividing cells that can be observed when examining the WT population indicates that cytokinesis is a process that can be accomplished in one hour and suggests that most of non-dividing cells are motile. Nevertheless, the long time spent to segregate dividing ∆ftsZ cells has no impact in the overall duration of the cell cycle in M. genitalium since doubling times are very similar in both strains, suggesting that the temporal control of the cell cycle in mycoplasmas is independent of the length of cytokinesis. This is in contrast with previous observations indicating the presence of specific checkpoints controlling the cell cycle of Caulobacter crescentus [START_REF] Hottes | DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus[END_REF][START_REF] Jensen | Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus[END_REF][START_REF] Shen | Architecture and inherent robustness of a bacterial cell-cycle control system[END_REF]. Alternatively, the time between two consecutive division rounds in M. genitalium may be long enough to provide a significant buffering capacity in the case of a delay in the cytokinesis, as has been observed in the ∆ftsZ cells.

We have provided evidence that mutations rendering non-adherent cells might be lethal in the ∆ftsZ strain. This was demonstrated by quantifying the appearance of viable non- may detach from the main cell body of M. pneumoniae cells deficient for P41 [START_REF] Hasselbring | Proteins P24 and P41 function in the regulation of terminal-organelle development and gliding motility in Mycoplasma pneumoniae[END_REF].

Mycoplasmas lack genes coding for Min and SlmA or Noc proteins. These proteins, especially the Min group, have a very important role as negative effectors of FtsZ polymerization and are essential for the establishment of cell polarity and the correct placement of the division site at midcell. SlmA and Noc proteins provide a second mechanism, the nucleoid occlusion system, to prevent the formation of septa over nucleoids [START_REF] Rothfield | Spatial control of bacterial division-site placement[END_REF]. On the other hand, M. genitalium strains with deletions in mg191 or mg192 lose the terminal organelle and exhibit a high frequency of cells of variable size and cells with a multilobed or a pleomorphic appearance, indicating the presence of defects in their cell division process [START_REF] Burgos | Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development[END_REF].

Preliminary work also indicates the existence of dramatic changes in the electrondense core ultrastructure of the cells from those strains, thus suggesting a role for cytoskeleton in the cell division of M. genitalium (Burgos et al., unpublished data). Because the cytoskeleton is the most notable polar component of this microorganism, this structure may also be involved in the placement of the Z-ring in WT cells. Therefore, the presence of a cytoskeleton in many mycoplasma species can also be seen as an opportunity to undertake many of the functions of cell division proteins missing in mycoplasmas.

One open question finally arises. If ftsZ is not essential for cell division in mycoplasma, why is this gene still present in the streamlined genome of M. genitalium? As is shown here, dividing ∆ftsZ cells remain fixed at the same location for extended periods of time.

Such lengthy time in the absence of movement may be detrimental for the in vivo survival of this mycoplasma, in this way providing a selective pressure against the loss of this gene. However, alternative explanations may also be possible, since dividing cells of M. pneumoniae, a related respiratory mucosal pathogen, can remain non-motile for several hours (Hasselbring et al., 2006a) despite the presence of a functional ftsZ.

We suggest an alternative possibility, related to the intracellular residence of M.

genitalium, also favoring the conservation of this gene. Soon after the internalization of M. genitalium inside eukaryotic host cells, mycoplasmas appear enclosed in vacuoles, many of them with a perinuclear location [START_REF] Jensen | Intracellular location of Mycoplasma genitalium in cultured Vero cells as demonstrated by electron microscopy[END_REF][START_REF] Mcgowin | Intracellular Mycoplasma genitalium infection of human vaginal and cervical epithelial cells elicits distinct patterns of inflammatory cytokine secretion and provides a possible survival niche against macrophage-mediated killing[END_REF][START_REF] Mernaugh | Properties of adhering and nonadhering populations of Mycoplasma genitalium[END_REF]. Since one of the prerequisites for gliding motility is the presence of a solid surface, it is plausible to think that the gliding force can be ineffective to support cell division in internalized mycoplasmas. In this scenario, ftsZ should be essential for the intracellular survival of M. genitalium. Fortunately, this hypothesis is fully testable and may reveal a new role for ftsZ as a virulence factor of mycoplasmas.

EXPERIMENTAL PROCEDURES

Bacterial strains and growth conditions. Escherichia coli strain XL-1 Blue was grown at 37 ºC in 2YT broth or LB agar plates containing 75 µg ml -1 ampicillin, 40 µg ml -1 X-Gal and 24 µg ml -1 Isopropyl-beta-thio galactopyranoside (IPTG) when needed. The M. genitalium G-37 WT strain was grown in SP-4 medium [START_REF] Tully | Enhanced isolation of Mycoplasma pneumoniae from throat washings with a newly-modified culture medium[END_REF] at 37 ºC under 5% CO2 in tissue culture flasks (TPP). To select the p∆ftsZTet M. genitalium transformant cells, plates were supplemented with 2 µg ml -1 tetracycline. To select pTnGftsZ complemented cells (TnGftsZ strain), plates were supplemented with 2 µg ml -1 tetracycline and 100 µg ml -1 gentamicin. S1 andS2. The purification of PCR products and digested fragments from agarose gels was achieved using the E.Z.N.A. Gel

Extraction Kit (Omega BIO-TEK).

Molecular cloning.

A 1-kb PCR fragment encompassing the upstream region of mg224 was obtained by using primers 5'BE∆ftsZ and 3'BE∆ftsZ, which contain the PstI and EcoRI restriction sites, respectively, at their 5' ends. Another 1-kb PCR fragment encompassing the downstream region of mg224 was obtained by using primers 5'BD∆ftsZ and 3'BD∆ftsZ that contain, at their ends, the BamHI and ClaI restriction sites, respectively. Both PCR fragments were cloned into EcoRV-digested pBE (Pich et al., 2006b), excised with the corresponding enzymes (Roche), and ligated together with a 2-kb BamHI-EcoRI fragment encompassing the tetM438 selectable marker (Pich et al., 2006b) and a PstI-ClaI digested pBSKII(+) (Invitrogen). This ligation mixture resulted in the p∆ftsZTet vector that was used to transform M. genitalium G-37 and obtain the mg224 null mutant (∆ftsZ). To construct the pTnGftsZ plasmid, a 1.1-kb PCR fragment encompassing the mg224 gene was first obtained by using primers 5'TnftsZ and 3'TnftsZ. Primer 5'TnftsZ contains a PstI restriction site and the promoter sequence of M. genitalium mg438 ORF (Pich et al., 2006b). Primer 3'TnftsZ contains a EcoRI resctiction site at its 5' end. The PCR fragment was cloned into EcoRV-digested pBE, excised with EcoRI-PstI enzymes, and ligated to the pMTnGm EcoRI-PstI digested vector (Pich et al., 2006b). The resulting vector was used to transform the ∆ftsZ strain as described below. Transformation of M. genitalium. Transformation of M. genitalium was performed by electroporation using 30 µg of DNA as previously described (Pich et al., 2006b).

Southern blotting and PCR assays. Genomic DNA of p∆ftsZTet transformants were digested with KpnI enzyme and hybridized as previously described (Pich et al., 2006b) using probe S1, which was a 1-kb fragment obtained by digesting p∆ftsZTet with PstI/EcoRI restriction enzymes (Fig. 1A). Genomic DNAs of ∆ftsZ and WT were digested with EcoRV and hybridized using probe S2, which was a 1.1-kb fragment encomprissing ftsZ amplified by PCR using 5'ftsZ and 3'ftsZ primers (Fig. 1A and Table S1). The ∆ftsZ mutants were also checked by PCR. The positive control was performed by amplifying a 1-kb PCR fragment encompassing the upstream region of mg224 by using primers 5'BE∆ftsZ and 3'BE∆ftsZ (Table S1) and WT or ∆ftsZ genomic DNAs as a template. To check the presence of ∆ftsZ mutants, a 1.1-kb fragment of ftsZ was amplified using 5'ftsZ and 3'ftsZ primers (Table S1) and genomic DNA of WT or ∆ftsZ as a template (Fig. 1C). Genomic DNAs of four TnGftsZ mutants were digested with EcoRV and hybridized using a probe of the gentamicin gene (data not shown). PCR products were sequenced to confirm the identity of the amplified fragments (data not shown).

RNA manipulations.

Total RNA from 20 ml of mid-log phase cultures of M.

genitalium G-37 and ∆ftsZTet mutants was extracted by using the RNAaqueous kit (Ambion). For RT-PCR assays, total RNA was treated with DNase I (New England Biolabs), and retrotranscribed using the SuperScript first-strand synthesis system kit (Invitrogen) and the 3'BDKOftsZ primer. The cDNA was amplified by PCR using the 5'RTmg225 and 3'BDKOftsZ primers, obtaining a fragment of 450 bp (Fig. 1D).

Electron microscopy. Samples were analyzed by TEM as previously described (Pich et al., 2008). Samples were analyzed by SEM according to the following procedure. Midlog phase cells grown in Lab-Tek chamber slides (Nunc) were washed three times with phosphate-buffered saline (PBS) and fixed with 1% glutaraldehyde for 1 h. Samples were washed three times with PBS and then dehydrated sequentially with 30%, 50%, 70%, 90% and 100% ethanol for 10 min each. Immediately, samples were critical point dried (K850 critical point drier; Emitech Ashfort; United Kingdom) and sputter coated with 20 nm thin gold layer. Samples were observed using a Hitachi S-570 (Tokyo, Japan) microscope. About 500 cells from each strain were analyzed to determine the frequency of cells in the different stages. The percentages of morphologies showing a terminal organelle at both poles of mycoplasma cells (Fig. 4A, stages 3 and 4) were used to estimate the length of cytokinesis. This length was calculated as the product of the frequency of dividing cells by the doubling time derived from the slopes of growth curves (see below).

Microcinematography. Samples with different cell densities were prepared as previously described (Pich et al., 2006a). Cell movement was examined at 37 °C using a

Nikon Quantitative plastic binding assay. Frozen stocks of the WT and ∆ftsZ cells were diluted in 1 ml of SP-4 medium to give a final concentration of approximately 10 7 CFU ml -1 . The binding assay was performed as previously described [START_REF] Burgos | Functional analysis of the Mycoplasma genitalium MG312 protein reveals a specific requirement of the MG312 N-terminal domain for gliding motility[END_REF].

Haemadsorption (HA) activity. For qualitative assessment of the HA, colonies grown in SP4 plates were flooded with 2 ml of human erythrocytes diluted (1:50) in PBS and incubated for 1 h at 37 °C. Plates were subsequently washed three times with PBS and observed using a LeicaMZFLIII microscope. Pictures were taken using a LeicaDC500 camera connected to the microscope.

Growth curves. To compare the growth kinetics of WT and ∆ftsZ cells, two independent experiments were performed using 48 cultures of 1 ml inoculated with approximately 10 7 CFU ml -1 and grown in 24 well TPP plates at 37 ºC. ATP content from six wells corresponding to each one of the different growth intervals was measured using the ATP Bioluminescence Assay Kit HS II (Roche). Measuring ATP instead of turbidity is considered the most proper method to estimate mycoplasma cell mass [START_REF] Lluch-Senar | A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium[END_REF][START_REF] Stemke | Measurement of Mollicute Growth bt ATP-Dependent Luminometry[END_REF]. Briefly, 1 ml of lysis reagent buffer (Roche) was added to each well and 50 µl samples were then placed in Wallac B&W isoplate polystyrene plates (Perkin Elmer). The ATP standard curve was constructed in the range of 10 -6 to 10 -12 M. Standards and samples were measured by duplicate after the automated injection of 50 µl of luciferase reagent in a Victor III luminometer (Perkin Elmer) using SP-4 medium as blank. ATP concentrations were calculated from a log-log plot of the standard curve data. The slopes corresponding to the exponential phase of growth were estimated from the linear regression of ATP values obtained from cultures grown for 0 h, 16 h, 24 h and 39 h. Mass doubling time of WT and ∆ftsZ was calculated from the slopes of the growth curves.

Obtaining and detecting non-adherent mutants. To detect spontaneous, non adherent phase variants, WT, ∆ftsZ and TnGftsZ strains were grown in 25 cm 2 flasks with 5 ml of SP-4 medium inoculated with approximately 10 7 CFU ml -1 . After ∼80 h the cultures were scraped off from the flasks and 2-ml samples of the suspensions were centrifuged to harvest cells, which were stored at -80 ºC for further DNA extraction. Successive passages were also performed in 25 cm 2 cell culture flasks with 5 ml of SP-4 that were inoculated with 50 µl of the cell suspension from the previous passage. The remaining of the cell suspension was passed through a 0.45 µm low binding protein filter (Millipore) and 300 µl samples of serial dilutions were spread on SP-4 plates, which were incubated 8 days at 37 °C. Plates showing 400-500 colonies (about 1,500 colonies per passage) were tested for haemadsorption as described above.

Quantitative PCR. The quantitative assay to detect the presence of recombinant chromosomes bearing deletions in the MgPa operon was performed using the LightCycler-FastStart DNA Master SYBR Green I (Roche) and the LightCycler.2 instrument (Roche). WT and ∆ftsZ genomic DNAs were used for two single-step PCR reactions using the R3-5' and R5-3' primers (Table S1) and primers mg281-5' and mg281-3'. R3-5' and R5-3' primers can detect the presence of a 558-bp DNA fragment (R3) resulting from the recombination between the MgPa operon and MgPa island VI (Fig. 5D) both in WT and ∆ftsZ genomes. Primers mg281-5' and mg281-3' amplify a standards (6.05×10 13 molecules ml -1 for pBE-R3 and 4.13×10 12 molecules ml -1 for pBE-T281; Table S2) were prepared in DEPC-H 2 O, and 2 µl of each was used for the calibration curve in the amplification reaction. The calibration curves, obtained by quantification of the serial standard dilutions, were plotted in the dynamic range from 1.21×10 4 to 12 molecules ml -1 for pBE-R3 and from 8.26×10 6 to 8.26×10 2 molecules ml -1 for pBE-T281. The concentration of the unknown-titer samples was obtained by interpolation on the calibration curve suggested by the software. A negative water control was added in each run to avoid overestimation of the specific product due to primer-dimer formation. Finally, the PCR amplification products were checked by agarose gel electrophoresis. Data in Fig. 5C (Pich et al., 2006b).

pBE-BEftsZ pBE derivative with a 1-kb fragment of the upstream region of the ftsZ gene cloned in the EcoRV site.

pBE-BDftsZ pBE derivative with a 1-kb fragment of the downstream region of the ftsZ gene cloned in the EcoRV site.

MTnTetM438

Plasmid used to obtain the tetracycline resistance gene (Pich et al., 2006b).

p∆ftsZTet Plasmid used to obtain the ∆ftsZ mutant.

pMTnGm Plasmid used to obtain pMTnGftsZ (Pich et al., 2006b). 3'BDKOftsZ (Table S1). Additional restriction sites used for cloning: PstI (P), EcoRI (E1), ClaI (C) and BamHI (B). 1 of the main text. SE: standard error. A ++ symbol indicates that all (∆ftsZ) or most (WT and TnGftsZ) colonies in the haemadsorption assay were found to be evenly covered with erythrocytes (Fig. 5A).

  . Therefore, if gliding motility is essential for cell division in the absence of ftsZ, no phase variants should be detected among cells of the ∆ftsZ strain.

  number of recombinant chromosomes, indicating that cells bearing a deletion in the MgPa operon do not accumulate along the serial passages and mutations rendering non-adherent cells are lethal in the ∆ftsZ strain.

  mycoplasma genomes has revealed the presence of few genes orthologous to those involved in cell division of most cell-walled bacteria. Although genes involved in the segregation of the peptidoglycan layer are obviously useless in mycoplasmas, it is perplexing to see how these microorganisms can divide by binary fission in the absence of several genes crucial for the Z-ring architecture. Moreover, the absence of these genes is difficult to explain, since their function cannot be rescued simply by uploading external cell resources, as is the case of genes involved in metabolism, and suggests the existence of alternative mechanisms in the cell division of these microorganisms. The previous isolation of a ftsZ transposon generated mutant prompted us to characterize the role of this gene and the encoded protein in M.

  adherent phase variants and chromosomes with deletions in the MgPa operon along serial culture passages. In these experiments, the frequency of non-adherent phase variants was very low in the first passages of WT cultures. This result can be explained by the fact that only collected cells growing attached to the plastic surface of culture flasks from routine cultures of M. genitalium WT cells were collected, thus avoiding the accumulation of non-adherent cells in the laboratory stocks. Since both adherent and non-adherent cells were used to start the next serial passages, the frequency of nonadherent cells progressively increased, reaching values close to 3%. This frequency is high enough to allow the detection of phase variants among the ∆ftsZ cell population if such non-adherent cells are viable. In the same way, recombinant chromosomes bearing deletions in the MgPa operon do not accumulate in the successive serial passages, supporting that HA -phase variants are not viable in ∆ftsZ cultures. Furthermore, SEM images suggest that the long tails in the mutant strain are produced as a consequence of the force generated by the gliding motility apparatus in the absence of FtsZ. Such tails become progressively longer and thinner until they are eventually broken. Taken together, these results strongly suggest that gliding motility is essential to segregate dividing ∆ftsZ cells. However, such broken tails have to be rapidly resealed to maintain cell integrity. Because ∆ftsZ cells grow at the same rate as WT cells, these data suggest that the mycoplasma cell membrane is very efficient when sealing such broken ends. A similar conclusion can be drawn from a previous work showing that terminal organelles

  Eclipse TE 2000-E microscope and images were captured with a Digital Sight DS-SMC Nikon camera controlled by NIS-Elements BR software. Particular movements of 600 individual cells from WT and mutant strains were analyzed to determine the frequency of motile cells during the observation period. Tracks from 50 individual motile cells corresponding to two min of observation and in six separate to determine the gliding velocity and gliding motility patterns.

  DNA fragment from mg281 (T281) to quantify the number of M. genitalium genomes in the PCR reactions. The amplified fragments were detected by emission of fluorophore SYBR green at the 485-nm wavelength and the quantitative determination was carried out during the exponential phase of amplification. Multiple real-time data acquisition and analysis of the samples and of the known standard serial dilutions were performed and analyzed by MyiQ software (Roche). Dilutions of the known-titer

pMTnGftsZ

  Figure 1. Engineering of ∆ ∆ ∆ ∆ftsZ mutants.

B)

  Southern blot to demonstrate the replacement of ftsZ by the tetM438 selectable marker. The 4.4-kb band was obtained by hybridizing the KpnI digestion of ∆ftsZ genomic DNA with probe S1.C) PCR detection of ftsZ. Lanes 1-3: PCR amplification using primers 5'ftsZ (P3) and 3'ftsZ (P4) and genomic DNAs of WT (lane 1) and the ∆ftsZ mutants obtained (lanes 2 and 3). Lanes 4-6: Positive controls of PCR amplifications using primers 5'BE∆ftsZ (P1) and 3'BE∆ftsZ (P2) and the same DNA templates used in lanes 1-3, respectively. D) RT-PCR of mg225 transcript in the WT and ∆ftsZ mutant. Total RNA from the M. genitalium G-37 (lane 2) and ∆ftsZ mutant (lane 4) was retrotranscribed using primer 3'BDKOftsZ (P6). The resulting cDNAs were amplified by PCR using primers 5'RTmg225 (P5) and 3'BDKOftsZ (P6), obtaining a fragment of 450 bp. The negative controls corresponding to the amplification by PCR of total RNAs from the WT and the ∆ftsZ mutant treated with DNase I are shown in lanes 1 and 3, respectively.

  Southern blot to detect the presence of ftsZ and related sequences in the genome of the ∆ftsZ mutant. EcoRV-digested genomic DNAs from WT and ∆ftsZ (lanes 1 and 2, respectively) were hybridized with probe S2 (lanes 3 and 4, respectively). A 10.8-kb band is readily detected in lane 3 and has the size expected according to the EcoRV sites shown in panel A. No bands were observed in lane 4 even after an extended detection time.

Figure 2 .

 2 Figure 2. Growth kinetics of WT and ∆ ∆ ∆ ∆ftsZ strains. Semi-logarithmic plot with the

Figure 3 .

 3 Figure 3. Colony and cell morphology of WT and ∆ ∆ ∆ ∆ftsZ strains.

Figure 4 .

 4 Figure 4. Stages in the cell division process of WT, ∆ ∆ ∆ ∆ftsZ and TnGftsZ strains.

Figure 5 .

 5 Figure 5. Quantification of non-adherent phase variants in serial culture passages.

  Frequency of chromosomes bearing deletions in the MgPa operon in serial cultures of WT (black squares) and ∆ftsZ (black dots). These recombinant genomes are the result of a double cross-over between R3 and R5 boxes of MgPa VI island and the homologous regions in the MgPa operon (see panel D). Frequencies are the average of five replicate Q-PCR measurements and bars represent the respective standard errors. D) Translocation of sequences from MgPa island VI to the MgPa operon in the M.genitalium genome. Schematic representation of a reversible, double cross-over event between the R3'-R3 and R5'-R5 boxes from the MgPa operon (bases 220,000 to 230,000) to MgPa island VI (bases 310,000 to 320,000). Boxes R1 to R6 refer to the DNA repetitive sequences of the MgPa operon, which are also found in MgPa island VI (R1' to R6') located 90 kb downstream. P1 and P2 indicate the position of the primers used for Q-PCR. A number of additional double-recombination events are also possible but are not included in the drawing for clarity. The location of the different MgPa islands in the M. genitalium genome can be found in[START_REF] Lluch-Senar | A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium[END_REF].

  Microcinematography showing gliding motility of WT cells. It is composed of 61 frames, each one taken at intervals of 2 s, and the resulting motion picture is shown at 10 frames s -1 . Labels 1-4 point to superimposed drawings of selected mycoplasma tracks (1, 3 and 4 show circular trajectories and 2 shows an erratic movement). Supplementary Movie S2. Microcinematography showing gliding motility of ∆ftsZ cells. It is composed of 61 frames, each one taken at intervals of 2 s, and the resulting motion picture is shown at 10 frames s -1 . Labels 1-4 point to superimposed drawings of selected mycoplasma tracks (1 shows an erratic movement and 2-4 show circular trajectories). Cells connected by a thin filament are indicated by white arrows and cells exhibiting long filamented tails by red arrows. Supplementary Figure S1. Complementation analyses of the ∆ftsZ mutant strain bearing a WT copy of the ftsZ allele (strain TnGftsZ). A) Representative colonies grown in culture dishes covered with SP-4 medium containing 0.5% low-melting-point agarose (bar, 250 µm). B) Frequency of HA -colonies generated in serial passages. C) Gliding parameters and haemadsorption properties. Data are compared with values from Table

  DNA genomic of M. genitalium was isolated by using the E.Z.N.A. Bacterial DNA Kit (Omega BIO-TEK). Plasmidic DNA was obtained by using the Fast Plasmid Mini Eppendorf Kit. All primers and plasmids used in this work are summarized in Tables

	DNA manipulations.
	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 1 .

 1 Sánchez-Chardi for his advice in transmission electron microscopy and Drs. J. L. 535 Arolas, A. Mozo and L. Strother for their helpful comments. 536 Parameters of WT and ∆ftsZ strains

	are derived from five replicate Q-PCR

Table S1 .

 S1 Primers used in this study 542

	Primer Name	Sequence (5'-3')
	5'BEKOftsZ CTGCAGAAAAGGTAGTTGTTGTTCTCGCTG
	3'BEKOftsZ GAATTCTTATTTAACCAAGCGTTGGAC
	5'BDKOftsZ GGATCCTTAATTTAATTTATCGTTTAGAATTGC
	3'BDKOftsZ ATCGATGAAGCAACTAAAGGGATAAAGAC
	5'ftsZ	ATGGATGAAAATGA
	3'ftsZ	TTAGTAGATTTGGTTTTGGTGCT
	5'TnftsZ	CTCTGCAGTAGTATTTAGAATTAATAAAGTATGGATGAAAATGA
	3'TnftsZ	GAATTCTTAGTAGATTTGGTTTTGGTGCT
	RTmg225	TCTTATGCAGGGGTTGAAGATATC
	R3-5' R3-3' mg281-5' mg281-3'	ACCGGACCTAACCTTGATAG GTAAAAATCTTATAAGAAGCAC GCATTTGACTTTTAATCAAAG GATTTCAACTTTACTTCTGC F o r
		P
		e
		e r
			R
			e
			v i e
			w

Table S2 .

 S2 Plasmids used in this study

	Plasmid	Source/Description
	pBE	pBSKII+ derivative with MCS removed and substituted by a single EcoRV site

ACKNOWLEDGEMENTS

This work was supported by grant BIO2007-67904-C02-01 from the MCYT (Ministerio de Ciencia y Tecnología, Spain) to E.Q. and by the Centre de Referència de R+D de Biotecnologia (Generalitat de Catalunya). E.Q. is the Chair Holder of a "Cátedra de Transferencia de Tecnología Parc de Recerca UAB-Grupo Santander". M.L-S. acknowledges a FPI predoctoral fellowship from the MCYT. We acknowledge A.


Glyn Williams

Richard Howard

Paul ; James

James Pj Frca

Howard Rf Frca

MD Williams Dg Frca

Dr Glyn David

Williams

analgesia infusion (PCA/NCA) increases analgesic efficacy in children with mucositis pain

Keywords: mucositis, ketamine, morphine, pain, infusion Pediatric Anesthesia Mucositis, Pain, Morphine, Ketamine, Infusion

The addition of ketamine to a morphine nurse or patient controlled analgesia infusion (PCA/NCA) increases analgesic efficacy in children with mucositis pain.

Background

Mucositis pain can be very difficult to control in some patients despite the use of parenteral opioids.

In our institution we have started adding low dose ketamine to the morphine NCA/PCA in these children in an effort to improve analgesic efficacy.

Methods/Materials

The records of all children receiving a morphine/ketamine PCA or NCA for mucositis pain in our institution from 1999 to 2007 were reviewed. At the time of treatment details of the analgesic management and consumption, pain scores and side-effects were prospectively recorded and then entered on to an electronic database. Ketamine was added at a concentration of 20 or 40mcg/kg/ml with our standard morphine NCA/PCA infusions and protocols being used.

Results

In 28 patients there was no difference between average morphine consumption in the 24 hours pre and post the addition of ketamine (33.1 (±10.7) vs 35.2 (±14.3) mcg/kg/hr, p=0.45) but in those with recorded pain scores (n=16) the median percentage of pain scores ≥ 4 was 48%(13-100%) pre ketamine versus 33%(0-82%) post ketamine (p=0.01). In all patients there was no change in the rates of nausea and vomiting and pruritis pre and post the addition of ketamine and no other significant side effects were reported. No difference was seen between those who had 20 or 40 mcg/kg/ml of ketamine added.

Conclusion

Introduction

Mucositis is defined as the painful inflammation and ulceration of the mucous membranes lining the digestive tract. It manifests by thinning of the mucosal lining, which may slough off, leading to erythema and ulceration. As the severity increases it is often associated with severe debilitating pain which can prevent talking, eating, swallowing and mouth opening [START_REF] Redding | Cancer therapy-related oral mucositis[END_REF][START_REF] Cheng | Palliation of oral mucositis symptoms in paediatric patients treated with cancer chemotherapy[END_REF]. Mucositis is usually seen as an adverse effect of chemotherapy and radiotherapy treatment for cancer. The incidence varies with the type and site of cancer and the method of treatment. Of adult patients receiving myeloablative chemotherapy for stem cell transplant 30 to 50 % suffer severe oral mucositis. This increases to over 60% if total body irradiation is part of the regime [START_REF] Redding | Cancer therapy-related oral mucositis[END_REF]. Children are thought to be at higher risk of developing oral mucositis and incidences from 52-80% have been reported following chemotherapy [START_REF] Bonnaure-Mallet | Oral Complications during treatment of malignant diseases in childhood: effects of toothbrushing[END_REF][START_REF] Childers | Oral complications in children with cancer[END_REF].

Mucositis due to chemotherapy typically begins 3 to 5 days after the start of therapy, peaks at 7 to 10 days, and slowly subsides over the next week. The most severe oral ulceration, and severe pain, tends to occur when the patient reaches the nadir of their white blood cell count. The pain can compromise the duration and success of disease management. Severe oral disease and pain makes adequate nutrition difficult and ulceration can predispose to infection with occasionally serious consequences in the immunosuppressed patient. Healing usually occurs within two to three weeks after chemotherapy is ended.

Mucositis pain is often very difficult to control. Meticulous oral hygiene remains the mainstay of treatment but in many cases systemic analgesia is also required. Mild analgesics such as Paracetamol and Non Steroidal Anti-Inflammatory Drugs are often inadequate or contraindicated and the oral and rectal routes of drug administration are usually unavailable. Severe mucositis pain is commonly for pain control for children with mucosistis has been previously described [START_REF] Dunbar | Use of patient-controlled analgesia for pain control for children receiving bone marrow transplant[END_REF][START_REF] Collins | Patient-controlled analgesia for mucositis pain in children: a three-period crossover study comparing morphine and hydromorphone[END_REF].

In our institution morphine nurse or patient controlled analgesia (NCA or PCA), alongside oral hygiene measures, has been the standard method of treatment for patients with severe mucositis pain.

In a proportion of patients, however, the provision of adequate pain relief has remained problematic.

In an effort to improve analgesic efficacy for this subgroup of patients low dose ketamine is now added to the morphine PCA or NCA. In this case series we would like to report our experience with children who received combined morphine-ketamine NCA/PCA for severe mucositis pain looking specifically at the effect on analgesic efficacy and the incidence of side effects.

Materials and Methods

The records of all children receiving a morphine/ketamine PCA or NCA for mucositis pain in our institution from 1999 to 2007 were retrospectively reviewed. At the time of treatment details of the analgesic management and consumption, pain scores and side-effects, including nausea and vomiting, pruritis, over sedation and hallucinations, were prospectively recorded and then entered on to an electronic database. All the children studied were given a demand-led morphine PCA or NCA infusion at a standard concentration of 20mcg/kg/ml. The decision to add ketamine was made on clinical grounds by a Pain Consultant if pain or the disease process were particularly severe, if adequate pain control was not achieved within normal NCA/PCA protocol limits or adequate pain relief was proving difficult to achieve and further disease progression was anticipated. Ketamine was added at a concentration of 20 or 40mcg/kg/ml with standard NCA/PCA protocols once again being used (Table 1). Whether 20 or 40mcg/kg/ml of ketamine was added was decided by the Pain Consultant at the time of the consultation using their judgement of the clinical situation, likely disease progression and potential analgesic requirement. Pain was assessed using self -report or behavioural observation using validated measures as appropriate (Faces Pain Scale -Revised or FLACC) [START_REF] Hicks | The Faces Pain Scale-Revised: toward a common metric in pediatric pain measurement[END_REF][START_REF] Manworren | Clincal validation of FLACC: preverbal patient pain scale[END_REF].

The PCA or NCA infusions were weaned as clinically indicated as the mucositis improved and were discontinued once pain could be managed effectively using oral analgesia. Statistical analysis was performed using t-tests and paired wilcoxon rank test for non parametric paired data using SPSS version 14. Differences were considered statistically significant if the p value was <0.05.

Results

A total of 35 patients with painful mucositis had ketamine added to their PCA/NCA regimen during the time period 1999 to 2007. Two could not be studied due to incomplete data collection. This left 33 patients available for study, 27 of whom had a NCA infusion and 6 a PCA infusion. Five children had the ketamine added at the commencement of the infusion. The patient characteristics are shown in Table 2. The primary diagnoses are shown in Table 3. Ketamine was added to all the infusions by day 6 of the infusion with the median being on day 4.

In 28 patients morphine consumption could be compared pre and post addition of ketamine. The results showed there was no statistical difference between average morphine consumption in the 24 hours pre and post the addition of ketamine (33.1 (±10.7) vs 35.2 (±14.3) mcg/kg/hr, p=0.45).

Seventeen patients had 20mcg/kg/ml ketamine added in this 24hr period compared with 11 patients who had 40mcg/kg/ml ketamine added. Comparison of these 2 groups showed no difference in morphine consumption between the two doses of ketamine.

Pain scores were available for 16 of these patients, as prior to 2002 pain scores were not entered into the database. The scores were recorded hourly and a score of 4 or greater was considered to be significant pain. Table 4 shows a comparison of the percentage of hourly pain scores greater than or equal to 4 in the 24 hours pre and post the introduction of ketamine. In all 16 patients there was a reduction in the percentage of pain scores greater than or equal to 4 after the introduction of ketamine.

The median percentage of scores ≥ 4 was 48%(13-100%) pre ketamine versus 33%(0-82%) post ketamine, p=0.01.

Of these sixteen patients 12 had 20mcg/kg/ml of ketamine added and 4 had 40mcg/kg/ml. In both groups there was a reduction in pain scores ≥4 pre and post the addition of ketamine but there was no difference in this effect between the 2 groups. Comparing infusion type in these sixteen patients showed that 12 had a NCA infusion and 4 a PCA infusion. Again both groups showed a reduction in pain scores ≥4 pre and post the addition of ketamine but once more there was no difference in this effect between the 2 groups.

In the 28 children who subsequently had ketamine added to the infusion the incidence of nausea and vomiting was 48% in the 24 hours preceding the addition of ketamine. This was reduced, though not significantly, to 41% after the introduction of ketamine (p=0.8). The incidence of pruritis in these 28 children was also similar pre and post addition of ketamine (38 and 34 % respectively, p=0.97). Again there was no difference in the incidence of these complications between the group who had 20mcg/kg/ml ketamine added and those who had 40mcg/kg/ml or between those who had a PCA or NCA infusion. The overall incidence of nausea and vomiting and pruritis in all 33 patients was 58% and 44% respectively In all the patients there were no reports of respiratory depression, sedation, vivid dreams, hallucinations, confusion or agitation at any point during their PCA/NCA, that could be attributed to the infusion, pre or post the addition of ketamine. One patient had the ketamine withdrawn after becoming acutely confused during a septic episode which required admission to PICU.

Discussion

The findings from our case series of children with chemotherapy induced mucositis suggest that the addition of ketamine to the routine morphine NCA or PCA regimen improves the efficacy of analgesia without increasing side-effects whilst the overall morphine consumption remains the same.

For each individual patient the course of the mucositis, and thus the severity of the symptoms, can be very different. The decision at which point to add ketamine to the infusion is made on clinical grounds and thus comparison between patients is difficult as the analgesic requirements vary enormously in terms of amount, time course and disease severity. Thus we decided to look at the time period around the addition of ketamine, thereby allowing each patient to act as their own control. With the decision to add ketamine being made on clinical grounds it is highly likely that the disease process and symptoms were deteriorating at that time with pain control becoming more problematic.

Looking at our data in this way showed that the average morphine consumption in the 24 hours pre and post the addition of ketamine did not change but the quality of the analgesia improved, as assessed by the patients` pain scores. The pain from mucositis is not always constant and can show troughs and peaks in severity throughout the day. Both the FLACC and Faces Pain Scale -Revised pain scores are 10 points scores [START_REF] Hicks | The Faces Pain Scale-Revised: toward a common metric in pediatric pain measurement[END_REF][START_REF] Manworren | Clincal validation of FLACC: preverbal patient pain scale[END_REF]. It was felt that a score of 4 or more constituted significant pain.

With pain being measured hourly the percentage of scores that were greater than or equal to 4 can be used as an overall guide to the efficacy of the analgesia within the 24 hour period.

As the pain associated with mucositis is often severe it is a frequent indication for the use of parenteral opioids [START_REF] Drake | Opioid rotation in children with cancer[END_REF]. Opioid requirements are often high and increase with the severity of the disease [START_REF] Dunbar | Use of patient-controlled analgesia for pain control for children receiving bone marrow transplant[END_REF]. In children aged between 12 and 18 years a comparison of morphine given by either PCA or continuous infusion showed similar efficacy but the morphine use and opioid related side-effects were lower in the PCA group [START_REF] Mackie | Adolescents use patient-controlled analgesia effectively for relief from prolonged oropharyngeal mucositis pain[END_REF]. Further studies have demonstrated the efficacy and safety of PCA or continuous infusion of opioids in children with mucositis over long periods of time and that, in terms of efficacy, the use of morphine and hydromorphone were comparable and superior to that of sufentanil [START_REF] Dunbar | Use of patient-controlled analgesia for pain control for children receiving bone marrow transplant[END_REF][START_REF] Collins | Patient-controlled analgesia for mucositis pain in children: a three-period crossover study comparing morphine and hydromorphone[END_REF]. Review of the adult literature for the treatment of mucositis pain with parenteral opioids also showed no difference in the efficacy of PCA versus continuous infusion but with the PCA patients demonstrating lower opioid requirements [START_REF] Worthington | Interventions for preventing oral mucositis for patients with cancer receiving treatment[END_REF]. The addition of low dose ketamine to opioids could theoretically improve analgesic efficacy for a number of reasons. Ketamine demonstrates anti-nociceptive properties in its own right. Using it as a co-analgesic with opioids may lead to improved analgesia in the same way as other regimens for multi-modal (or synergistic) analgesia. The decrease in opioid tolerance could potentially lead to opioid sparing effects and improved efficacy. Ketamine will also abate the induction of opioid related hyperalgesia.

In adults low dose ketamine is increasingly being used as an adjunct to opioids in the peri-operative period. Recent reviews suggest that addition of ketamine to peri-operative parenteral opioids decreases post-operative opioid consumption, improves analgesic efficacy and respiratory outcome with no increase in side-effects. Though when combined in a PCA the results are equivocal and the use of a single shot administration intra-operatively is insufficient to provide long lasting pain relief [START_REF] Subramaniam | Ketamine as adjuvant analgesic to opioids: a quantitative and qualitative systematic review[END_REF][START_REF] Sveticic | Safety of mixture of morphine with ketamine for postoperative patient-controlled analgesia: an audit with 1026 patients[END_REF]. A single dose of ketamine in the postoperative period has also been shown to rapidly and effectively treat severe pain that was incompletely relieved by morphine [START_REF] Weinbroum | A single small dose of postoperative ketamine provides rapid and sustained improvement in morphine analgesia in the presence of morphine-resistant pain[END_REF]. Low dose ketamine has also been shown to reduce opioid requirements as well have analgesic efficacy in its own right in adult patients with cancer related pain. Though, as the studies show little homogenicity in terms of design the authors state that more investigation is required to assess the potential benefits and sideeffects [START_REF] Bell | Ketamine as adjuvant to opioids for cancer pain. A qualitative systematic review[END_REF]. When added together in the same syringe morphine and ketamine have demonstrated both compatibility and stability [START_REF] Trissel | A Handbook on Injectable Drugs[END_REF].

Ketamine has been used to treat cancer related pain in children [START_REF] Finkel | Ketamine as an adjuvant for treatment of cancer pain in children and adolescents[END_REF]. The use of ketamine as an adjunct to opioids has been less well studied in children. Two systematic reviews which include limited paediatric data show that the addition of ketamine to opioids in the perioperative period is safe and efficacious. The role of PCA morphine and ketamine, however, remains unclear [START_REF] Subramaniam | Ketamine as adjuvant analgesic to opioids: a quantitative and qualitative systematic review[END_REF][START_REF] Elia | Ketamine and postoperative pain -a quantative and systematic review of randomised trials[END_REF]. Also no opioid sparing effect is seen with the use of a single shot administration intra-operatively [START_REF] Batra | Intraoperative small-dose ketamine does not reduce pain or analgesic consumption during perioperative opioid analgesia in children after tonsillectomy[END_REF][START_REF] Becke | Intraoperative low-dose S-ketamine has no preventive effects on postoperative pain and morphine consumption after major urological surgery in children[END_REF]. In children with cancer pain a ketamine infusion alongside opioids has been shown to increase analgesic efficacy, decrease opioid use and improve quality of life [START_REF] Tsui | Intravenous ketamine infusion as an adjuvant to morphine in a 2-year-old with severe cancer pain from metastatic neuroblastoma[END_REF][START_REF] Conway | Use of continuous intravenous ketamine for end-stage cancer pain in children[END_REF]. The use of ketamine only NCA and PCA regimens, without any concurrent opioid use, has been reported in children with inflammatory bowel disease and demonstrated both safety and analgesic efficacy [START_REF] White | Pain management in fulminating ulcerative colitis[END_REF]. Our results demonstrate an overall incidence of 58% for nausea and vomiting and 42% for pruritis associated with the morphine and ketamine infusion. This compares with rates of 60% and 36% for our patients with chemotherapy induced mucositis treated with a morphine only NCA or PCA (GOSH pain service database-personal communication). In addition, looking at the 24 hours pre and post the addition of ketamine, there was no increase in the incidence of side effects. In fact the rate of nausea and vomiting fell slightly (48 vs 41%) as did the incidence of pruritis (38 vs 34%). Reviews of adult literature have shown no increase in side-effects, including central nervous system effects, associated with the addition of low dose ketamine to opioids with some studies showing a decrease in the incidence of nausea and vomiting and pruritis [START_REF] Subramaniam | Ketamine as adjuvant analgesic to opioids: a quantitative and qualitative systematic review[END_REF][START_REF] Bell | Peri-operative ketamine for acute post-operative pain: a quantitative and qualitative systematic review[END_REF].

The incidence of side-effects in children receiving ketamine and opioids has not been well studied.

Intraoperative use of ketamine demonstrated no increase in psychomimetic effects or nausea and vomiting compared with control groups [START_REF] Batra | Intraoperative small-dose ketamine does not reduce pain or analgesic consumption during perioperative opioid analgesia in children after tonsillectomy[END_REF][START_REF] Dix | Double-blind randomized placebo-controlled trial of the effect of ketamine on postoperative morphine consumption in children following appendicectomy[END_REF][START_REF] Flaherty | Does ketamine or magnesium affect post-tonsillectomy pain in children?[END_REF]. In one study where ketamine was also given as a postoperative infusion, 4 out of 25 of the children in the ketamine infusion group had the infusion stopped due to lightheadedness or hallucinations [START_REF] Dix | Double-blind randomized placebo-controlled trial of the effect of ketamine on postoperative morphine consumption in children following appendicectomy[END_REF]. Though the infusion rate was 240mcg/kg/hr compared with our rates of 20 or 40 mcg/kg/hr. The symptoms resolved in all cases within 60 minutes of the infusion stopping. In children with a ketamine infusion for cancer pain there were no adverse effects reported [START_REF] Tsui | Intravenous ketamine infusion as an adjuvant to morphine in a 2-year-old with severe cancer pain from metastatic neuroblastoma[END_REF][START_REF] Conway | Use of continuous intravenous ketamine for end-stage cancer pain in children[END_REF].

Our results showed that there was no difference between adding 20 or 40mcg/kg/ml of ketmine to the morphine infusion in terms of morphine consumption, efficacy or complications. This was also shown to be the case when comparing infusion technique. The numbers, however, in each of these groups were small, especially in the 40mcg/kg/ml and PCA groups. Thus it is hard to comment on the clinical benefit of using the higher dose of ketamine or a particular infusion technique, especially at the outset of treatment with ketamine. This is an area that requires further study.

Pain associated with mucositis in children can be debilitating and very difficult to treat. Our case series of children with worsening mucositis shows that adding ketamine to morphine improves analgesic efficacy and could potentially be considered a beneficial development in the management of

  opioid analgesia given parenterally. The use of patient controlled opioid analgesia

6

 6 All relevant data was extracted from the electronic database and stored on an Excel spreadsheet.

N

  -methyl-D-aspartate (NMDA) receptors have a central role in pain transmission. Activation of these receptors via the release of glutamate from excitatory synapses plays an important part in central sensitisation and wind-up and the development of hyperalgesia and allodynia. NMDA receptor activation also decreases neuronal sensitivity to opioid receptor agonists. Ketamine is a noncompetative antagonist at NMDA receptors. It prevents the induction of central sensitisation and wind-up and the development of opioid tolerance. Ketamine also has agonist actions at spinal opioid receptors and at monoamine receptors in the descending inhibitory pathways.

  The decision to add ketamine must also be balanced against the possible adverse effects. Our data shows no increase in the incidence of side effects although due to the limited number of cases and the non-randomised nature of the study further research in this area is required.

1 Legends Table 1 .

 Legends1 NCA/PCA Infusion Protocols. GOSH Pain Control Service.

Table 2 .

 2 Patient Characteristics. Data are mean (range), median or number.

Table 3 .

 3 Primary diagnosis of patients

Table 4 .

 4 Percentage of hourly pain scores ≥ 4 for the twenty four hours before and after the addition of ketamine to the morphine NCA/PCA, infusion type and dose of ketamine in the 16 patients studied.Comparison of the median percentages shows a significant decrease in hourly pain scores post the addition of ketamine (48% vs 33%, p=0.01).

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	1

Table 1 .

 1 Up to 50kg = morphine 1mg/kg (+/-ketamine 1mg/kg or 2mg/kg) in 50mls 5% glucose

		Background (ml/hr)	Bolus (ml)	Lockout (mins)
	PCA	0 or 0.2	0.5 or 1	5 or 10
	NCA	0,0.2,0.5 or 1	0.5 or 1	20 or 30
	>50kg = morphine 50mg (+/-ketamine 50mg or 100mg) in 50mls 5% glucose
		Background (ml/hr)	Bolus (ml)	Lockout (mins)
	PCA	0	1 or 2	5 or 10
	NCA	0, 0.2, 0.5 or 1	1	20 or 30

Table 2 .

 2

	Sex (male/female)	20/13
	Age (yr)	5.1 (0.3-13.6)
	Weight (kg)	21.3 (8 -104)
	Infusion Duration (days)	16.8 (5 -32)
	Ketamine Added (day)	4 (0 -6)

Table 3 .

 3

	Primary diagnosis	No. of Patients
	Acute Lymphoblastic Leukaemia	8
	Acute Myeloid Leukaemia	5
	Haemophagocytic Lymphohistiocytosis	5
	Severe Combined Immunodeficiency	3
	Neuroblastoma	4
	Lymphoma	4
	Kostmanns Syndrome	1
	Behcets disease	1
	Osteopetrosis	1
	Primitive Neuroectodermal Tumour	1

Table 4

 4

	Patient Number	Pain scores≥4	Pain scores≥4	Infusion Technique	Ketamine Dose
				(BDG = Background)	(mcg/kg/ml)
		pre ketamine(%)	post ketamine(%)
	1	57	40	NCA + BGD	40
	2	50	33	NCA + BGD	20
	3	30	0	NCA + BGD	20
	4	40	33	NCA + BGD	40
	5	12	9	NCA + BGD	20
	6	11	0	PCA	20
	7	60	0	PCA	20
	8	100	82	NCA + BGD	40
	9	45	37	NCA + BGD	40
	10	67	33	NCA + BGD	20
	11	44	35	NCA + BGD	40
	12	57	33	PCA	40
	13	29	0	PCA = BGD	40
	14	14	13	NCA + BGD	20
	15	91	40	NCA + BGD	20
	16	52	41	NCA + BGD	20
		Median 48	Median 33
		Range 13-100	Range 0-82

Acknowledgements

The authors would like to thank Theresa Snelling and Justin Buys for their help in the management of the Pain Control Service Database.


Arnwald ; Choi

N Ahmad

D De Beer

METABOLIC CHANGES DURING MAJOR CRANIOFACIAL SURGERY

Keywords: craniofacial, metabolic, prospective

Metabolic changes during major craniofacial surgery (Prospective Observational Study of the Metabolic Status of Children Undergoing Major Craniofacial

Surgery)

The purpose of this study was to document the degree and duration of perioperative metabolic disturbance during major craniofacial surgery in children.

AIM

The aim was to quantify the degree and duration of perioperative metabolic disturbance and secondly to determine the relationship between the metabolic changes and the duration of surgery and total volume of blood and colloid given during surgery.

BACKGROUND These patients have the potential for massive blood loss and significant metabolic acidosis.

Routine perioperative monitoring includes the serial measurement of base deficit as a marker of metabolic disturbance.

METHODS/MATERIALS

All patients undergoing elective major craniofacial surgery were prospectively studied over a 10-month period. Base deficit from arterial blood gas analysis was measured at standardised intervals during the perioperative period. The duration of surgery and total volume of blood and colloid given intraoperatively were used as covariates in a multiple regression analysis.

RESULTS

Maximum recorded base deficit ranged from -3 to -20 (median -9). Median time taken to return to normal was 9.25h (range 0-18h). Median duration of significant base deficit was 3.8h (range 0-20h). surgery can be categorised according to whether they are undergoing procedures for surgical correction of single suture craniosynostosis and fronto-orbital remodelling or more extensive surgery including monobloc/fronto-facial advancement. They have the potential for massive blood loss and significant metabolic acidosis [START_REF] Soldin | Pediatric Reference Ranges[END_REF][START_REF] Stricker | [END_REF]. Routine perioperative monitoring includes base deficit analysis from serial arterial blood gas sampling. The current practice in this Trust is for this to be continued postoperatively for all patients, overnight on the high dependency unit.

The aim of the study was to quantify the degree and duration of metabolic disturbance associated with major craniofacial surgery using the current practice of clinical monitoring.

In addition, the relationship between the base deficit and both duration of surgery and total volume of blood and colloid given during surgery was analysed.

Patients undergoing monobloc/fronto-facial advancement were excluded from the study on the basis that they constitute a different surgical group and their perioperative care is managed differently. The primary outcome measure was base deficit from arterial blood gas analysis. This was used as a marker of metabolic disturbance and measured at standardised intervals during the perioperative period. A significant base deficit was defined as being 33% of the normal range less than the lower parameter, i.e. a base excess of less than -6 (normal reference range for base excess in children is -4 to +2) (1). Duration of surgery and total volume of blood and colloid given intraoperatively were used as covariates in a multiple regression analysis.

Preoperative data were collected, including age, gender, weight, ASA grade, significant past medical history, presence of associated syndrome, time of last oral intake before surgery, and laboratory results (including preoperative haemoglobin, platelet count, urea, creatinine and electrolytes). The type and amount of fluid administered during the intraoperative period were recorded.

The primary outcome measure, base deficit, was recorded from arterial blood gas analysis using the i-STAT® Portable Clinical Analyser. This was measured at standardised time intervals, from time of insertion of arterial cannula, hourly during surgery, at application of bandages, on arrival to the recovery area, and four-hourly on the high dependency unit until the patient was discharged to the general ward area or removal of arterial vascular access. Duration of surgery, as defined by time taken from "knife to skin" to application of bandages after completion of the surgical procedure, and the total volume of blood and colloid given intraoperatively were recorded as covariates for subsequent data analysis.

Other intra-and postoperative data which were documented included the type of surgical procedure, clinical observations at time of base deficit measurement (heart rate, mean arterial blood pressure, peripheral oxygen saturation, capillary refill time, core temperature, urine output, central venous pressure if monitored), volume and type of fluid given, and blood glucose at time of sampling.

Anaesthetic technique consisted of intravenous or gaseous induction followed by maintenance of general anaesthesia with a volatile inhalational agent.

A descriptive numerical and graphical data analysis was carried out using the Statistical Package for Social Science for Windows version 15 (SPSS Inc., Chicago, IL, USA). Multiple regression analysis was performed on the primary outcome measure to determine the relationship between the magnitude of metabolic acidosis and the covariates. In order to detect a moderate correlation (0.5) between the outcome measure and covariates, a sample size of at least 25 patients was required (power 80%, p < 0.05). A total of 28 patients were recruited during the study period. Out of these 28 patients, 19 (68%) analysed were male. The number of patients assessed as ASA 1 was 9 (32%), 13 (46%) as ASA 2, and 6 (21%) as ASA 3. There were four patients who were recorded as having Crouzon syndrome, two with Saethre-Chotzen, one with Apert, and one with Pfeiffer syndrome. The median age at surgery was 19m (range 4m-17y). There were 18 (64%) patients under 2 years of age at time of surgery. Weight of patients ranged from 6.2 to 54.0 kg (median 11.9). At time of surgery, 21 (75%) patients weighed 15 kg or less.

A total of 13 (46%) of the surgical procedures were classified as fronto-orbital remodelling for single suture craniosynostosis. The remaining procedures included sagittal biplication, and posterior and biparietal vault expansion. The median operative time was 3.1h (range 1.2-

7.8h).

The median value for base deficit (BD) at the start of surgery was -4 (range -7 to +1) while for maximum recorded perioperative base deficit it was -9 (range -20 to -3). The capillary refill time was noted to be normal (<2s) at the time of each arterial blood sampling. The median time from commencement of surgery to maximum base deficit was 3.5h (range 0-11.5h) while the time taken to return to a normal level, defined as a BD ≥-6, was 9.25h (range 0-18h). The median duration of significant base deficit, as defined by a BD < -6, was 3.8h (range 0-20h). A median total volume of 55ml.kg -1 (range 10-148ml.kg -1 ) of blood and colloid was administered intraoperatively. Only one patient did not receive blood intraoperatively, but a blood transfusion was administered in the immediate postoperative recovery period. Craniofacial surgery is performed in children in order to correct deformities of the cranial bones, orbits and midface. This type of surgery is often performed at an early age and is associated with prolonged operating times, extensive operative exposure, and significant heat and blood loss [START_REF] Thompson | Assessment and treatment of craniosynostosis[END_REF][START_REF] Tuncbilek | Blood loss and transfusion rates during repair of craniofacial deformities[END_REF]. Estimation of intraoperative blood loss in patients undergoing craniosynostosis repair is often inaccurate and significantly underestimates actual blood loss [START_REF] Faberowski | Blood loss and transfusion practice in the perioperative management of craniosynostosis repair[END_REF][START_REF] Mallory | Anaesthetic management in facial bipartition surgery: the experience of one centre[END_REF]. Almost all patients undergoing repair of craniofacial deformities will need a blood transfusion with potential for massive blood transfusion (4). One objective of intraoperative monitoring is to guide fluid replacement improve perioperative outcome and reduce adverse events [START_REF] Papworth | Intraoperative monitoring during vascular surgery[END_REF].

The development of a metabolic acidosis is common in patients undergoing extensive surgery involving significant blood loss [START_REF] Marietta | Pathophysiology of bleeding in surgery[END_REF]. This may be the result of a combination of blood loss and electrolyte shifts following the administration of large amounts of crystalloid, colloid and blood products. The base deficit, an indicator of metabolic acidosis, has been shown to correlate with the severity of haemorrhagic shock in patients with multiple trauma, and can be used in quantifying the response to haemorrhage in trauma or non-trauma patients [START_REF] Rixen | Bench-to-bedside review: Oxygen debt and its metabolic correlates as quantifiers of the severity of haemorrhagic and post-traumatic shock[END_REF]. Furthermore, haemodynamic and haemorrhagic complications of surgery have been shown to be associated with a low base deficit value and an increased length of stay in the intensive care unit [START_REF] Hugot | Base deficit in immediate postoperative period of coronary surgery with cardiopulmonary bypass and length of stay in intensive care unit[END_REF]. A recent study by Stricker et al shows that a significant metabolic acidosis occurs within both the intra and postoperative period during major craniofacial surgery [START_REF] Stricker | [END_REF].

However our study describes the metabolic changes throughout the time course during the perioperative period. This study recorded a wide variation in degree and duration of metabolic disturbance in the intra-and postoperative period. A severe metabolic derangement developed in 11 (39%) of the patients, with a measured base deficit of ≤ -10. For five patients (18%) the duration of significant acidosis lasted for over 10 hours. In 23 patients (79%), the maximum base deficit occurred after the end of surgery with the majority (14) having a subsequent period of significant metabolic acidosis during the postoperative care in the recovery area and on the high dependency unit. One patient developed ongoing severe metabolic acidosis along with clinical deterioration and required subsequent emergency management and transfer to intensive care. Intravenous bicarbonate was given on an individual basis alongside clinical assessment. No other patients received intravenous bicarbonate. Three patients still had a significant base deficit following overnight high dependency care, but did not need a higher level of critical care. The majority of patients (86%) had a measured base deficit in the normal range following the overnight care on the high dependency unit. Arterial blood gas sampling was taken at hourly intervals intraoperatively and 4 hourly postoperatively. This limits the accuracy of our study in assessing the maximum base deficit and return to normal range.

The total volume of blood and colloid given intraoperatively was used as a surrogate measurement of blood loss. The assumption is made that all patients were maintained in a euvolaemic status at the time of each arterial blood gas measurement, as all recorded clinical observations of capillary refill time were normal (< 2 seconds) at the time of sampling. Any coagulopathic derangements were also corrected intraoperatively as guided by haematological laboratory results. There was a statistically significant relationship between maximum base deficit and total volume of blood and colloid given intraoperatively. However the degree of metabolic disturbance does not appear to be related to the duration of surgery. This suggests that it is difficult to predict which patients will develop a greater or persistent metabolic acidosis by length of surgical procedure alone. Intravenous propofol was not used to maintain anaesthesia and therefore it is unlikely that any metabolic disturbance was due to a propofol infusion syndrome.

We designed the study protocol around current practice without additional resources or intervention apart from the data collection sheets. Chloride and lactate are not routinely measured with the iSTAT® cartridges in current use, so we based our assessment of metabolic change without these parameters. We are therefore unable to define a more specific nature of the metabolic disturbance, such as hyperchloraemic acidosis, lactic acidosis or ketoacidosis. Although perioperative acidosis is suggestive of end organ hypoperfusion it is important to distinguish ongoing tissue hypoxia from hyperchloraemic metabolic acidosis secondary to fluid infusion because they should be managed differently. The correct interpretation of an abnormal base deficit in a surgical patient may be difficult to disentangle because of its multifactorial nature. A physicochemical approach to analysis of acid-base disturbance may be helpful in distinguishing the aetiologies [START_REF] Stephens | Optimising intraoperative Fluid Therapy[END_REF]. Of direct relevance to this study is the work by Brill et al which suggests that base deficit does not predict mortality when secondary to hyperchloraemic acidosis [START_REF] Brill | Base deficit does not predict mortality when secondary to hyperchloremic acidosis[END_REF]. Future studies which incorporate additional laboratory analysis would be needed to evaluate the base deficit in more detail.

Patients undergoing monobloc/fronto-facial advancement surgical procedures, which are carried out less frequently in our institution, may be investigated as a separate group in future studies in order to compare blood loss and fluid requirements and metabolic changes. This study is the first documented data on metabolic changes during major craniofacial surgery. It did not intend to analyse the aetiology of the changes nor specify a threshold of base deficit that would trigger a decision to admit to a high dependency unit. As with many biochemical measurements, the data should be looked at on an individual basis in conjunction with data trends and clinical assessment of each patient.

Our data from this study has confirmed the presence of marked metabolic disturbance, in both degree and duration, during major elective paediatric craniofacial procedures. This is associated with considerable intraoperative blood loss and volume replacement, and as a useful marker of physiological derangement, it can remain significant for several hours into the postoperative period. Biochemical data should not be viewed without reference to clinical review. All patients undergoing major craniofacial surgery in our Trust continue to have monitored overnight postoperative care on the high dependency unit.

  craniofacial surgery develop a varying degree of perioperative metabolic acidosis persisting for several hours. The maximum base deficit appears to be related to the amount of intraoperative blood loss and replacement rather than duration of surgery. As it is difficult to predict the extent and duration of metabolic acidosis for an individual patient, this study confirmed our current practice that all patients should be admitted to a neurosurgical high dependency unit postoperatively for overnight monitoring. Hospital is one of several Trusts which carry out complex elective surgery for paediatric craniofacial abnormalities. Children undergoing major craniofacial

  Committee exemption was attained on the basis that no additional interventions or changes to standard perioperative care would be instituted. All patients undergoing elective major craniofacial surgery (fronto-orbital remodelling, sagittal biplication, posterior and biparietal vault expansion, but excluding monobloc distraction/fronto-facial advancement) were prospectively studied over a 10-month period.

  of variance (ANOVA) was used with weight as a dependent variable to allow for the size of each patient. Using maximum base deficit as the independent variable and total volume of blood and colloid given intraoperatively and duration of surgery as dependent variables, there was a statistically significant relationship between maximum base deficit and volume of fluid (p = 0.02, correlation coefficient = -0.835, r 2 = 0.466) but no significant relationship with duration of surgery (p = 0.58, correlation coefficient = 0.193, r 2 = 0.293).

ACKNOWLEDGEMENTS

We are extremely grateful for the advice and support from Professor T.Cole (Department of Paediatric Epidemiology and Biostatistics, Institute of Child Health).