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1 Department of Molecular, Cellular and Animal Biology, University of Camerino, Camerino, Italy
2 Struttura Complessa di Microbiologia, Azienda Ospedaliera di Perugia, Perugia, Italy
Correspondence
Luca Agostino Vitali
luca.vitali{at}unicam.it
Received 10 September 2007
Accepted 26 November 2007
Abbreviations: CIP, ciprofloxacin; CLI, clindamycin; CVC, central venous catheter; ERY, erythromycin; GEN, gentamicin; MRSA, meticillin-resistant Staphylococcus aureus; MSSA, meticillin-susceptible Staphylococcus aureus; OXA, oxacillin; PEN, penicillin; QD, quinupristin/dalfopristin; RIF, rifampicin; TEL, telithromycin; TET, tetracycline.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Among intravascular devices, the use of central venous catheters (CVCs) is frequently followed by both local and systemic complications, including septic thrombophlebitis, endocarditis, metastatic infections and bacteraemias (Maki & Mermel, 1998). In particular, it is estimated that over 80 % of all catheter-related bloodstream infections are associated with CVCs (van Belkum, 2000).
MRSA strains can be investigated by various typing schemes. They appear to be closely related genetically (Enright et al., 2000). Many molecular epidemiology studies clearly indicate that the massive geographical spread of MRSA results from the dissemination of relatively few highly epidemic clones (Oliveira et al., 2001).
The first stage in the emergence of MRSA is acquisition of the mecA gene and associated mec DNA by meticillin-susceptible S. aureus (MSSA), and their integration into its chromosome. The mecA gene encodes an extra penicillin-binding protein PBP 2a or PBP 2' that allows cell wall synthesis to continue despite inactivation of native penicillin-binding proteins. There are five genetic classes of the mec gene complex, each consisting of an intact copy of mecA, a copy of IS431mec and, when present, complete or truncated mec regulatory genes mecI and mecR1 (Hiramatsu et al., 2001). The mec gene complex is carried on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec). This locus also contains a cassette chromosome recombinase (ccr) complex, consisting of the ccr genes ccrA and ccrB in combination (ccrAB) or the ccrC gene alone, in addition to the adjacent ORFs (Ito et al., 2004). The rest of the SCCmec element, which lies outside the ccr and mec complex, is known as the junkyard or J region. It may contain various antibiotic resistance genes. To date, five types of SCCmec element (I–V) and a number of subvariants have been characterized (Grundmann et al., 2006).
The origin of these elements, as well as the number of times that these foreign pieces of DNA have entered the S. aureus species, and the mechanisms of their acquisition remain substantially unknown and are the subject of lively scientific debate (Deurenberg et al., 2007). Recent studies of the evolutionary history of MRSA suggest multiple introductions of the five SCCmec elements into MSSA strains with the same sequence type, indicating that horizontal transfer of mec genes is relatively frequent within S. aureus (Lim et al., 2003; Oliveira et al., 2002; Robinson & Enright, 2003).
In this work, we analysed a group of S. aureus strains isolated from central intravenous catheters in patients presenting clinical signs of bacteraemia and positive blood cultures. The antibiotic resistance profile was determined, along with the ability to form a biofilm on inert surfaces, followed by PCR identification of the genetic determinants of resistance/biofilm production and SmaI macrorestriction followed by PFGE. The SCCmec elements of the identified MSRA strains were subsequently defined and related to the other investigated characteristics.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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15 c.f.u.) (Mermel et al., 2001). Two independent isolates from different samples taken from the same patient were obtained and identified using a Vitek 2 system instrument. Thirty-seven single S. aureus isolates were collected. S. epidermidis ATCC 35984 (complete genome sequence: GenBank accession no. NC_002976) was used as a biofilm-positive reference strain together with S. epidermidis 1457 (Mack et al., 1994). The S. epidermidis transposon mutant 1457-M11 was used as a biofilm-negative strain (Mack et al., 1994). Staphylococcal pandemic reference strains used in SmaI macrorestriction profiling by PFGE and in the determination of the SCCmec type were (each strain is reported as ‘name of strain-SCCmec type-multilocus sequence typing sequence type’): COL-I-st250, HPV107-IA-st247, BK2464-II-st5, HUSA304-III-st239, HSJ216-IIIA-st239 (Oliveira et al., 2002), PER34-IA-st250 (Dominguez et al., 1994), JP1-II-st5 (Aires de Sousa et al., 2000), HU25-IIIA-st239 (Teixeira et al., 1995), PL72-IV-st5 (Leski et al., 1998), BM18-IV-st5 (Roberts et al., 1998), BAR2529-V-st8 (Kreiswirth et al., 1993) and UK13136-I-250 (Crisostomo et al., 2001). Isolated bacteria and reference strains were stored at –80 °C. Antibiotic susceptibility. The determination of MICs was performed in accordance with the Clinical Laboratory Standards Institute guidelines (CLSI, 2005). Penicillin (PEN), oxacillin (OXA), erythromycin (ERY), tetracycline (TET), clindamycin (CLI), telithromycin (TEL), gentamicin (GEN), ciprofloxacin (CIP), quinupristin/dalfopristin (QD) and rifampicin (RIF) (Oxoid) were tested by the disc diffusion method. The Etest and microdilution methods were used for linezolid (AB Biodisk) and vancomycin (Sigma), respectively.
Biofilm formation. The ability to form a biofilm was evaluated using the crystal violet staining test according to a previously described protocol (Petrelli et al., 2006). This method measures the crystal violet stain retained by cultured bacterial cells adhering to the bottom of microtitre plate wells after repetitive cycles of washing. All strains that showed a low biofilm formation capacity with the above described crystal violet method were retested following the protocol described by Christensen et al. (1985).
PCR screening of the genetic determinants of antibiotic resistance, adhesion and biofilm formation. All amplification reactions were prepared in a 25 µl volume containing: 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 1.25 mM MgCl2, 100 µM each dATP, dCTP, dGTP and dTTP, 1 µM each oligonucleotide primer, 1 U Taq polymerase and 200 ng template DNA. All strains were investigated to detect the presence of genes associated with the screened antibiotic resistances, namely: blaZ (PEN resistance); mecA (OXA resistance); tetK, tetM, tetL and</italic> tetO</italic> (TET resistance); ermA, ermB, ermC, msrA, vatA, vatB, vatC, vgaA and vgaB (ERY, CLI, TEL and QD resistance); and aac(6')–aph(2'') (GEN resistance).
Oligonucleotide primer sequences for the detection of antibiotic resistance-associated and biofilm-associated genes are reported in Table 1
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Macrorestriction and PFGE.
The general conditions for macrorestriction with SmaI and PFGE have been described by Chung et al. (2000). The S. aureus ATCC 29213, ATCC 25923, ATCC 43300 and RN4220 strains were used as references. The obtained PFGE profiles were analysed using 1D image analysis software (Kodak) and compared directly. For every pair, the Dice coefficient (DC) was calculated (DC=number of shared electrophoretic bandsx2x100/overall number of electrophoretic bands in the two samples). Strains with a DC 80 % were considered to be strictly related, whilst those with a 60 % 
DC 80 % were classified as related, and subsequent nomenclature was assigned according to the rules reported by Ripa et al. (2001).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Of the 37 strains, 89.2 % were resistant to PEN, whilst 43.2 % were resistant to OXA and were classified as MRSA. Nearly half of the strains were resistant to both ERY and CLI, and 88.2 % of this latter subgroup were not susceptible to TEL. Importantly, the two ERY/CLI-resistant and ketolide-susceptible strains showed an inducibly D+ resistance pattern towards CLI, with a D-shaped zone of inhibition around the CLI disc (Steward et al., 2005). One isolate out of thirty-seven was resistant to QD (strain SA019). TET was inactive towards 10.8 % of the isolates and partially active against one (strain SA105). Resistance towards members of the aminoglycosides (GEN), quinolones (CIP) and rifamycines (RIF) was recorded in 37.8, 35.1 and 21.6 % of the isolates, respectively. All isolates were susceptible to LZD (MIC50=1.0 mg l–1; MIC90=2.0 mg l–1) and vancomycin (MIC50=0.5 mg l–1; MIC90=1.0 mg l–1).
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, the population presented a full correlation between the presence of the blaZ genetic trait and resistance to PEN, whilst 2/16 MRSA isolates were mecA negative. The presence of specific resistance genes among the ERY-resistant group was also evaluated. All isolates were negative for the msrA gene, which encodes the staphylococcal ERY efflux system. Among ERY resistance methylase genes, the most prevalent was ermA, which was detected alone in half of the erm-positive subpopulation, whilst ermB was recorded in only one isolate. ermA/ermC association occurred in three isolates, whilst the ermC gene alone was detectable in four. All ermA and ermA/ermC genotypes were associated with the constitutive macrolide, lincosamide and streptogramin B phenotype (resistance to both ERY and CLI), except for isolate SA134, which had intermediate resistance and was susceptible to the macrolide and the lincosamide. In the group that was positive for ermC alone (n=4), half of the isolates showed a constitutive phenotype, whilst the other half were ERY-resistant and CLI-susceptible, expressing a D+ phenotype (D-shaped zone of inhibition around the CLI disc). tetK, tetM, tetL and tetO were evaluated to determine the TET resistance genotypic distribution. None of the tested isolates were positive for tetL or tetO. Two TET-resistant isolates (SA088 and SA102) carried tetK, whilst SA134 was positive for tetM. SA019 contained both tetK and tetM. The intermediate resistance phenotype did not harbour any Tet determinants. All GEN-resistant isolates and the susceptible SA102 isolate harboured aac(6')–aph(2''). QD-resistant SA019 was positive for ermA and vgaA.
This analysis of 37 S. aureus isolates from CVC-related infections confirmed the well-established multiresistant character of staphylococci in the hospital setting. We found that PEN, OXA, GEN and ERY co-resistance was very common and its genetic basis could be defined with the exception of a GEN-susceptible isolate containing the aac(6')–aph(2'') gene and two MRSAs that were negative for the mecA gene. The latter occurrence has been observed previously in S. aureus and may be due, at least in part, to β-lactamase overexpression (Martineau et al., 2000). SA019 was the single isolate that was resistant to all tested antibiotics, including QD. Its resistance to streptogramins was due to a methylase gene (ermA) and a specific gene encoding an ATP-binding protein (vgaA).
Only two MSSA were susceptible to all tested antibiotics. In general, MSSA did not show an extended multiresistance. For instance, 10/21 (47.6 %) MSSA were resistant only to one antibiotic (i.e. PEN or ERY), and 6/21 (28.6 %) were resistant to two (i.e. PEN/RIF, PEN/TET or TET/ERY).
We found a significant correlation between OXA resistance and resistance to ERY, CLI, TEL, GEN and CIP. This evidence is in line with previous reports and confirms the generally held idea that MRSA is resistant to several drugs and other toxic elements (e.g. cadmium) (Oliveira & Lencastre, 2002). In particular, CIP is a fluoroquinolone, which is among the most commonly prescribed classes of antimicrobial drugs in both the hospital and the community. By the early 1990s, many MRSA isolates from clinical specimens were found to be resistant to CIP. Moreover, several recent investigations have offered preliminary evidence suggesting that fluoroquinolones themselves may actually predispose patients to infection with or carriage of MRSA (Crowcroft et al., 1999; Harnett et al., 1991).
Biofilm formation
The presence of the icaA and icaD genes was assessed by PCR. One major group was observed (Table 2). Of the 37 clinical isolates that were analysed, 94.6 % contained both icaA and icaD. These genes were not found in two isolates. To obtain a semi-quantitative estimate of biofilm formation level, a crystal violet staining methodology was used. This method measures the amount of crystal violet stain retained by cultured bacterial cells adhering to the bottom of microtitre plate wells after repetitive cycles of washing. The mean intra-plate coefficient of variation was calculated by including an in-plate control strain; this variability was below 15 % (90 % degree of confidence). The mean inter-plate coefficient of variation for each isolate was below 20 % (90 % degree of confidence). All but three isolates presented a mean A540 value above 0.12, which is the lower limit for a biofilm-forming strain (Christensen et al., 1985). In the icaAD-positive (icaAD+) group, the mean A540 value was 0.734 (SD=0.906). Among the icaAD+ isolates, SA057, SA116 and SA146 were the only biofilm-negative samples.
Two isolates were found to be icaAD-negative (icaAD–). The mean A540 value of these isolates was 0.540 (SD=0.040 units).
Using PCR, we screened our population for the presence of both pls (an anti-adhesive factor) and atl (a factor involved in initial adherence) genes, and evaluated their individual and possible synergistic contributions to the formation of biofilms in the context of an icaAD+ or icaAD– background. Their presence did not correlate significantly with biofilm-forming capacity or the inability of the isolates to adhere to plastic surfaces.
Phenotypic and genotypic analyses of the ability of isolates to form biofilms in vitro revealed that the isolates achieved a good general level of growth on polystyrene surfaces. This finding is consistent with the site of isolation (i.e. catheters). However, a high degree of variability in biofilm formation capacity was recorded among isolates possessing the icaAD operon, ranging from strong biofilm producers to those that could not adhere to the plastic surface of the microtitre wells. In contrast, icaAD– isolates were able to produce a certain level of biofilm, well above that measured for at least half of the icaAD+ isolates. The ica operon is considered to be one of the main genetic determinants of the accumulation phase during biofilm formation and its detection has been suggested as a tool for discriminating invasive from contaminating strains in clinical specimens (Arciola et al., 2002; Vandecasteele et al., 2003). Our observations showed, however, that icaAD+, weak biofilm-producing S. aureus can be isolated from CVCs. The same conclusion has also been reached by other authors, supporting the general statement that the ica operon is not suitable as a discriminatory test for the capacity of invasiveness, at least in the case of S. epidermidis (Rohde et al., 2004). On the one hand, isolates lacking the ica operon are able to sustain growth as a biofilm; however, some of the ica operon-positive isolates produce an equivalent or smaller amount of biofilm than ica operon-negative isolates. This finding is rather novel in the specific case of S. aureus isolated from CVCs, but is not surprising, as it has already been reported for coagulase-negative and coagulase-positive staphylococci (Cafiso et al., 2004; de Silva et al., 2002). Moreover, the SD calculated for the values of biofilm mass formed by icaAD+ group is high. This is an indication of great variability and, at least in terms of biofilm mass production, seems to confirm that the presence of genes encoding the polysaccharide intercellular adhesin does not constitute an absolute determinant of biofilm-formation ability.
Genetic relatedness and SCCmec element analysis
Molecular analysis of SmaI-digested DNA resulted in 31 distinct PFGE types (Table 3), of which 45.2 % were recorded in only one isolate (one-strain type, ost). The remaining patterns fell into six type clusters (A–F). As expected, PFGE analysis closely correlated with the presence of mecA and meticillin resistance (Table 3). In just one PFGE class (type cluster D), both meticillin-susceptible (mecA-negative) and -resistant (mecA-positive) isolates were present. Type cluster B was related to archaic clones (COL and UK13136 strains), whilst the PFGE profile of the other strains did not match any of the pandemic clones used as references. Significant relatedness between PFGE patterns and other resistance phenotypes/genotypes was not observed.
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). Strain SA019 was classified as SCCmec type IIIA-ccrAB3, but its PFGE profile did not match any of the IIIA-positive Brazilian pandemic clonal type strains used as references, namely HU25 and HSJ216. The remaining four mecA-positive strains were found to possess a SCCmec element that was only partially or not at all related to any of the previously described types; these strains were therefore classified as unknown type (Table 3). Strains SA097 and SA098 showed PCR amplification profiles consistent with type I, but with positive amplification of the cif2 region and negative amplification of the coding region for the pls gene (Table 2). This variation was additionally confirmed by the impossibility of clustering the PFGE profile of these strains with those of the type I archaic clones. Nevertheless, these strains conserved a type I basic structure as revealed by the positive amplification of the ccrAB1 complex. Strain SA075 was positive for all of the elements that characterize the SCCmec type IA with the additional amplification of the mecI portion. The ccr analysis confirmed the relationship with type I, but also indicated a relationship with the type III ccr (Table 3). Analysis of SA040, SA053 and SA057 did not result in classification of the SCCmec elements harboured by these strains. SA040 was positive for cif2, but not for the dcs region that is characteristic of the type I SCCmec, and it contained both ccrAB1 and ccrAB2. cif2 was associated with pT181, ccrAB1 and ccrAB2 in SA053, whilst SA057 would be compatible with a type IV SCCmec with the exception that it was positive for the mecI region. Moreover, in the latter strain, none of the tested ccr complexes was amplified, even when the universal pair of primers, which amplifies all types of known ccr complexes, was used (Table 3). The striking finding of this study is the degree of genetic variation within SCCmec in MRSA isolated from catheter-associated infections. At the genomic level, this evidence was also confirmed by SmaI macrorestriction profiling. Two strains appeared to be variants of the archaic clone MRSA-I. They lacked a portion of the L-C region mapping 5' of the type I SCCmec. Shore et al. (2005) described a similar SCCmec I type element in nine strains isolated in Ireland. This region contains pls, which is not thought to be an important part of SCCmec (Ito et al., 2004), but it has been implicated in biofilm formation in staphylococci (Savolainen et al., 2001). However, the adhesion properties of these SCCmecI-pls– strains did not differ significantly from either the parental SCCmecI or from the other MRSA and MSSA strains. One strain showed a type IA variant that was positive for amplification of the mecI region and the ccrAB complex of both types I and III. This observation could be explained either by a genetic rearrangement that occurred between genetic cassettes belonging to the two different classes or by the transfer of a new SCCmec element variant from another species. Lastly, three strains presented genetic organizations of SCCmec that could not be related to any of the known variants of this element, confirming that there have been major changes in the dominant clonal types in this group of strains from catheter-related infections in hospitalized patients. This genetic individuality was also confirmed by PFGE typing and supports the great flexibility of MRSA to evolve in addition to its general diffusion as pandemic clones. To the best of our knowledge, this is the first study reporting such variability in a set of strains isolated from CVCs. In this context, the high frequency of isolation of atypical MRSA from device-related sources of infection raises concerns over the possible importance of growth on surfaces for the selection of new variants. This hypothesis is supported by the observation reported by Prunier et al. (2003) that some bacterial populations growing as biofilms present a high proportion of hypermutable strains, and by the fact that the exchange of genetic material between bacteria, and consequent genetic rearrangement by recombination, is greatly favoured in the particular environment created by a biofilm (Molin & Tolker-Nielsen, 2003).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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