1 Antibiotic Resistance Monitoring and Reference Laboratory and 2 the Respiratory and Systemic Infection Laboratory, Specialist and Reference Microbiology Division, Health Protection Agency, Colindale, London NW9 5HT; 3 Birmingham Health Protection Agency Laboratory, Birmingham B9 5SS; 4 Division of Immunity & Infection, Medical School, University of Birmingham, Birmingham B15 2TT, UK
Received 12 June 2003; returned 7 August 2003; revised 5 September 2003; accepted 9 September 2003
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Abstract |
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Methods: Isolates were characterized by multilocus sequence typing (MLST), PFGE, and sequencing of parC and gyrA. Relevant clinical data were sought.
Results: Forty-eight isolates were received from nine laboratories in England, but 35 (73%) were from one laboratory in Birmingham, and were mostly from elderly patients receiving ofloxacin or ciprofloxacin for respiratory infections. There were two quinolone resistance phenotypes, with ciprofloxacin, moxifloxacin and gemifloxacin MICs of 832, 0.51 and 0.1250.25 mg/L, and 64256, 416 and 14 mg/L, respectively. Each of three isolates from the former group had mutations in parC, whereas each of nine isolates from the more resistant group had mutations in both parC and gyrA. Several also had increased quinolone efflux. Typing of 27 quinolone-resistant isolates showed that eight were indistinguishable from the epidemic Spain9V-3 (ST156) clone, while the remainder belonged to a novel but related type (ST609), that differed from Spain9V-3 at 2/7 alleles (2 bp changes in aroE and 1 bp change in gdh). Both MLST types were represented among isolates with high- and low-level quinolone resistance. Three of five serotype 9V isolates from Birmingham, with reduced susceptibility to penicillin and erythromycin, and ciprofloxacin MICs of 12 mg/L, belonged to MLST type ST609, while another was indistinguishable from the Spain9V-3 clone. Review of records of 32 patients from Birmingham indicated that some isolates were nosocomial, whereas others were acquired in the community.
Conclusions: In the late 1990s, a quinolone-resistant strain, clonally related to Spain9V-3, emerged in England, principally in Birmingham.
Keywords: Streptococcus pneumoniae, MLST, quinolone resistance
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Introduction |
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The treatment of multiresistant pneumococcal infections is problematic, and there is a need for new agents, particularly those suitable for oral administration. Several fluoroquinolones with enhanced anti-pneumococcal activity and favourable pharmacokinetics were developed during the 1990s,6 and are now included among the agents recommended by several expert panels for patients with pneumonia, particularly where penicillin- or macrolide-resistant Streptococcus pneumoniae is known or suspected.710 Surveys show that quinolone resistance in pneumococci is rare in many countries, although data from Canada indicate the beginnings of a rising trend.11 Exceptionally, the prevalence of resistance to fluoroquinolones is already high in pneumococci from Hong Kong.12
In an earlier investigation, we evaluated moxifloxacin against a collection of referred pneumococci with elevated resistance to ciprofloxacin.13 In assembling the panel of isolates, it was noted that half were phenotypically similar, belonging to serotype 9V and showing reduced susceptibility to penicillin and erythromycin. Most of these isolates had been referred, over several years, from Birmingham Public Health Laboratory (PHL) (now Birmingham Health Protection Agency Laboratory). This raised the possibility that a quinolone-resistant strain might have become established in Birmingham. We present here a detailed molecular study to determine the clonal relationship of these and other isolates with the same resistance phenotype, referred from Birmingham and elsewhere in the UK.
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Materials and methods |
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Isolates for study were selected by searching the databases of the Central Public Health Laboratory, to identify pneumococci referred during 19972001 that had the same phenotype as the isolates studied previously, namely serotype 9V, penicillin MICs 1 mg/L, erythromycin MICs
2 mg/L and ciprofloxacin MICs
8 mg/L (the highest concentration routinely tested). A further five isolates referred from Birmingham PHL in 1997, which were phenotypically similar, except for having ciprofloxacin MICs of 12 mg/L, were also included. All the isolates were recovered from frozen stocks, and had their serotype and antibiogram re-confirmed. The Pneumococcal Molecular Epidemiology Network reference strain for the Spain9V-3 clone, S. pneumoniae ATCC 700671, served as a reference for MLST and PFGE typing.5
Patient data
Clinical details of the patients whose isolates were referred from Birmingham PHL were reviewed retrospectively from case notes, hospital infection control records and the hospital admissions database. Antibiotic prescribing data for the Thoracic Medicine Department of Birmingham Heartlands Hospital during April 1996April 2001 were obtained from pharmacy records. The Local Ethics Committee Chairman advised that this review of patients records did not require formal Ethics Committee approval.
Susceptibility tests
MICs were determined in air on Diagnostic Sensitivity Test agar (Oxoid, Basingstoke, UK) containing 5% lysed horse blood (TCS Microbiology, Buckingham, UK), as described previously.13 Isolates were categorized as susceptible or resistant using breakpoints recommended by the British Society for Antimicrobial Chemotherapy.14
Serotyping
Serotypes of pneumococci were determined by slide agglutination with capsular typing sera.15,16
Multilocus sequence typing
MLST was performed as described previously.17 In brief, internal fragments of the genes aroE, gdh, gki, recP, spi, xpt and ddl were amplified by PCR, and the sequences of each amplicon were determined in both the forward and reverse directions, using the Dye Terminator Cycle Sequencing Quick Start Kit (Beckman-Coulter, Fullerton, CA, USA) and analysed on a CEQ 2000 DNA Analysis System (Beckman-Coulter). The sequences were then compared with those of all the recognized alleles of each gene listed in the pneumococcal MLST website database (http://spneumoniae.mlst.net). Sequences that were identical to those of a known allele were assigned the same allele number, whereas novel sequences were submitted, with the original CEQ sequence trace files, to the database curator, who assigned new allele numbers. The resulting allelic number profiles were checked against the database to find the corresponding sequence type (ST) numbers. Allelic profiles not represented were submitted to the database curator for assignment of new ST numbers. The MLST allele profile data were analysed using BioNumerics software (Applied Maths, Kortrijk, Belgium) with a categorical data analysis method. Trees were drawn using the unweighted pair group method with arithmetic means (UPGMA).
Pulsed-field gel electrophoresis
Low-melting-point agarose gel blocks containing bacteria were prepared, and the cells were lysed following the protocol of McEllistrem et al.,18 with modifications. Specifically, the DNA was digested with 20 U of SmaI overnight at 30°C and electrophoresed on a 1% agarose gel in a CHEF DR II PFGE tank, with an initial switch time of 0.5 s, rising to 25 s over 28 h. Photographs of the gels were analysed using BioNumerics software, and clustering of PFGE patterns was performed by Pearson correlation, allowing a UPGMA tree to be drawn.
Sequencing of the quinolone resistance determining regions of gyrA and parC
Mutations in the quinolone resistance determining regions of gyrA and parC were investigated for 12 isolates. PCR amplifications were performed as previously described,19 using the primer pairs VGA3 and VGA4 for parC, and M0363 and M4271 for gyrA, and amplicons were sequenced as above. Sequences were analysed using BioNumerics and Genebase software (Applied Maths), and compared with reference sequences for a quinolone-susceptible isolate (gyrA, GenBank accession no. AJ005815; parC, GenBank accession no. Z67739).
Determination of efflux phenotype
To ascertain if efflux played a role in reduced susceptibility to quinolones, MICs of norfloxacin and ethidium bromide were determined in the presence and absence of the efflux pump inhibitor reserpine (10 mg/L).20 Efflux-mediated resistance was inferred from resistance to norfloxacin (MIC > 8 mg/L) and ethidium bromide (MIC > 16 mg/L), together with a four-fold or greater reduction in the MIC of norfloxacin in the presence of reserpine. The efflux phenotype was confirmed by transforming the ethidium bromide resistance into S. pneumoniae strains R6 and R6(pmrA::cat), as previously described.21,22 Transformants were selected on Columbia blood agar containing ethidium bromide at 4 mg/L, and resistance was confirmed by subculture onto fresh medium containing ethidium bromide at 6 mg/L.
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Results |
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Interrogation of the reference laboratory databases revealed that 48 isolates received during 19972000 met the inclusion criteria, being of serotype 9V, with ciprofloxacin MICs 8 mg/L and penicillin MICs
1 mg/L. All except one also had low-level resistance to erythromycin (MICs 28 mg/L). Thirty-five of the isolates were received from Birmingham PHL. Of these, 32 were from respiratory samples (30 sputa and two bronchial traps) and three were from blood cultures. The other 13 isolates were from eight other hospitals. One hospital was located in Sutton Coldfield,
7 miles north of Birming-ham, five were in areas (Stoke-on-Trent, Shrewsbury, Chester, Merseyside, and the Wirral) 5095 miles to the north or north-west of Birmingham, and the other two were in London and Portsmouth, both over 100 miles away.
When MICs of quinolones were re-determined using an extended range of concentrations, two quinolone resistance phenotypes were observed among the isolates. Eighteen isolates, designated as having low-level quinolone resistance (LLQR) were resistant to ciprofloxacin (MICs 832 mg/L), and had moxifloxacin and gemifloxacin MICs that were either on or two-fold below their breakpoints (MICs 0.51 and 0.1250.25 mg/L, respectively). The remaining 30 isolates were designated as having high-level quinolone resistance (HLQR), with ciprofloxacin, moxifloxacin and gemifloxacin MICs of 64256, 416 and 14 mg/L, respectively. Nine of the 35 isolates from Birmingham PHL and 8/13 isolates from elsewhere, had the LLQR phenotype, whereas 26/35 Birmingham isolates and 5/13 isolates from elsewhere had the HLQR phenotype.
Patient details
The 35 isolates from Birmingham PHL were from 33 patients (20 males, 13 females), all aged 50 years (four were aged 5059 years, six were 6069 years, 17 were 7079 years and six were >80 years old). Clinical records were available for 32 of these patients (31 inpatients and one outpatient), and showed that most had acute respiratory tract infections (17 were diagnosed with pneumonia and seven with exacerbations of airway disease) and/or underlying chronic obstructive pulmonary disease (n = 26). Recurrent admissions to the hospital were common, with 16 source patients (50%) having had an admission in the preceding 3 months. Sixteen inpatients had isolates collected more than 1 week after admission, and 12 had a preceding negative sputum culture, implying nosocomial infection. Potential epidemiological links were observed for some patients, suggesting nosocomial spread of the resistant pneumococci. A cluster of five cases occurred in succession during late November 1997early January 1998 on one ward, together with two on another ward (Table 1). Six of these seven isolates were collected on or after the third day of admission (range 340 days), the exception being one that was collected on the day of admission. The source patient for this latter isolate had recently been discharged from the other affected ward. MLST (see below) showed that the isolates from six of these seven patients belonged to ST609 (the other isolate belonged to ST156), which is consistent with interpatient spread of infection.
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Twenty-seven of the patients whose isolates were referred from Birmingham PHL were under the care of physicians at one Birmingham hospital. In 1997, the hospitals antibiotic policy for the first-line treatment of pneumonia was changed from amoxicillin and erythromycin to ofloxacin, with or without penicillin. Figure 1 shows the Respiratory Directorates increased use of ofloxacin, and to a lesser extent, ciprofloxacin and penicillin V, during this period, with a concurrent reduction in amoxicillin use. Review of the records of 32 patients showed that quinolones had been prescribed in 20 (63%), penicillin in 12 (38%) and amoxicillin in three (9%), during the 3 months before isolation. Of the 20 patients prescribed a quinolone, 18 received ofloxacin and two received ciprofloxacin. Unfortunately, clinical records were not available for the patients from the other hospitals.
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Thirty-two serotype 9V isolates were examined by MLST. These were selected so as to include quinolone-susceptible (n = 5), LLQR (n =7) and HLQR (n = 7) phenotypes from Birmingham, and all 13 resistant isolates from the other referring hospitals (Table 2).
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Pulsed-field gel electrophoresis
DNA from 12 representative isolates and S. pneumoniae ATCC 700671 (the reference strain of the Spain9V-3 clone) was subjected to PFGE analysis. The 12 isolates were selected to include representatives from Birmingham PHL and four other centres, the three MLST types observed, quinolone-susceptible, LLQR and HLQR phenotypes, and isolates with different gyrA and parC mutations (see below). All the tested isolates showed possible or close relationships to the Spain9V-3 reference strain (Figure 2); three gave identical patterns over at least 13 fragments, while the remainder showed between two and four band differences from the reference isolate, and thus would be classified as closely or possibly related to Spain9V-3, based on Tenovers criteria.23
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The PFGE and MLST data were combined, using BioNumerics software, to produce the relatedness dendrogram shown in Figure 3. This analysis confirmed that the penicillin- and quinolone-resistant serotype 9V pneumococci from Birmingham and elsewhere in the UK are closely related to the Spain9V-3 (ST156) clone, but clustered into two broad groups, corresponding to ST156 and its newly described two-locus variant, ST609, respectively. Isolates with LLQR and HLQR were indistinguishable by PFGE and MLST, suggesting sequential acquisition of quinolone resistance mutations within each of the related lineages.
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Amino acid substitutions, inferred from DNA sequence changes for three isolates with LLQR and nine isolates with HLQR, are shown in Table 3. Among the LLQR isolates (all ST609), there were no changes to GyrA, but all three showed changes at amino acids 79 and 137 in ParC. In two LLQR isolates from Birmingham PHL, the change at position 79 was SerTyr, whereas in an LLQR isolate from Merseyside, the change was Ser
Phe. In all three isolates, the change at position 137 was Lys
Asn. Each of nine isolates of the HLQR phenotype had mutations in both parC and gyrA. In eight of these, GyrA had the amino acid substitution Ser-83
Phe, with Ser-83
Val in the other one. In seven of the nine, the mutations in parC caused the amino acid substitution Ser-79
Tyr, whereas the change Ser-79
Phe was seen in the other two. All nine isolates had other mutations in parC causing the change Lys-137
Asn (Table 3).
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Thirteen of the 48 isolates, including representatives from Birmingham and six other centres, showed an efflux phenotype. Efflux was variably present in isolates of the LLQR (8/18) and HLQR (5/30) phenotypes, and was also variably present in isolates of ST609 and ST156. The efflux phenotype from a selection of nine isolates was transformed successfully into both R6 and R6(pmrA::cat).
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Discussion |
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MLST analysis indicated that a clone of quinolone-resistant S. pneumoniae, designated ST609, emerged and persisted in the patient catchment area for Birmingham PHL for several years. This conclusion was reinforced by PFGE analysis, which confirmed that representative isolates were highly related. The quinolone-resistant clone was not, however, confined to the patient catchment area of Birmingham PHL, as isolates of the same ST were also received from three other hospitals. One was in Sutton Coldfield, which is only about 7 miles north of Birmingham PHL, whereas the other two were in Merseyside and Shrewsbury, both within 95 miles. On the basis of MLST and PFGE, ST609 appeared closely related to the Spain9V-3 clone (ST156), which was also foundwith quinolone-resistant representativesin Birmingham and elsewhere. ST156 and ST609 differed at two house-keeping loci, but only by one or two nucleotides in each gene. Thus, it seems reasonable to regard the members of ST156 and ST609 as comprising a single clonal complex. A further observation supporting this view is that whereas the international clone Spain9V-3 is usually susceptible to erythromycin,5 all the isolates of ST156 and ST609 bar one reported here were erythromycin resistant. It is interesting that quinolone-resistant isolates of ST156 and ST609 were both seen in Birmingham and other regions of the UK, suggesting that this clonal complex might have a propensity to develop resistance to quinolones. In this context, it is noteworthy that quinolone-resistant isolates from France and Northern Ireland, apparently belonging to the Spain9V-3 clone, have recently been reported by others.26
Nosocomial spread of quinolone-resistant pneumococci has been documented previously in the USA.27 There was also strong evidence of nosocomial infection in this study, although the high re-admission rates of some of the Birmingham patients makes it difficult to determine where acquisition occurred. On the other hand, there was also evidence of community spread, with resistant isolates obtained within 1 day of admission from three inpatientsnone of whom had been hospitalized in the preceding 3 monthsas well as from one outpatient.
Quinolone-susceptible isolates of ST609 were present in Birmingham in 1997, and possibly before then. The earliest variants with the LLQR phenotype also date from 1997, and those with the HLQR phenotype from 1998. The latter organisms appear to have emerged as a result of sequential mutations in parC and gyrA. The association of changes in parC in pneumococci with low-level quinolone resistance, and additional changes in gyrA in isolates with a higher level of quinolone resistance, are consistent with previous reports, several of which describe the same mutations as found here.28,29 Whilst efflux only has a small effect on ciprofloxacin MICs it may help cells resist low levels of antibiotic, and thereby allow the accumulation of further resistance mutations.20,21 However, the function of such pumps requires cellular energy, and once a cell has developed high-level resistance due to topoisomerase mutations, there is no need for the expenditure of this energy and the selective advantage of maintaining the efflux pump may be lost. It is not surprising to find the efflux phenotype to be less frequent in pneumococci with HLQR. We were able to transform the efflux phenotype into a pneumococcus in which pmrA was insertionally inactivated. This is evidence that systems other than PmrA may be contributing to efflux. Such PmrA-independent efflux has been described previously in laboratory mutants21 and is not surprising since genome sequencing of pneumococci indicates a large number of putative multi-facilitator and ABC superfamily efflux pump genes.
Modelling studies suggest that the risk of an individual being colonized by pneumococci is independent of antibiotic exposure.30 When, however, individuals are colonized by pneumococci, the risk of these being antibiotic-resistant is related to antibiotic exposure. A multivariate case-control analysis previously identified chronic obstructive pulmonary disease and quinolone exposure as risk factors for acquisition of fluoroquinolone-resistant strains.31 Review of the clinical records for Birmingham patients from whom quinolone-resistant pneumococci of ST609 were obtained revealed that most had been prescribed ofloxacin or ciprofloxacin for lower respiratory tract infections. Although this use of quinolones is supported by some authorities and guidelines,710 these mostly recommend the use of newer quinolones such as moxifloxacin, which have enhanced anti-pneumococcal activity and a lower potential to select first- and second-step quinolone-resistant mutants. It is possible that the documented use of ofloxacin or ciprofloxacin, which have poorer anti-pneumococcal activity, provided the selective pressure for the emergence and persistence of the quinolone-resistant strains described here. However, we cannot discount the possibility that the use of penicillin or erythromycin, to which the isolates were also resistant, may have exerted an additional selective pressure, either in the hospital or community.
Spread of quinolone-resistant variants of the ST609 clone does not yet seem to have impacted significantly on the overall prevalence of quinolone resistance of S. pneumoniae in the UK,32 but the presence of this clone, related to the highly successful pandemic pneumococcal strain Spain9V-3, is a cause for concern. Continued surveillance, coupled with molecular characterization of isolates is clearly warranted to monitor further dissemination.
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Acknowledgements |
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Footnotes |
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Present address. University Hospital Birmingham NHS Trust, Queen Elizabeth Hospital, Birmingham B15 2TH, UK.
¶ Present address. Wells Healthcare, Speldhurst Place, Speldhurst Road, Tunbridge Wells TN4 0JB, UK.
Present address. Wells Healthcare, Speldhurst Place, Speldhurst Road, Tunbridge Wells TN4 0JB, UK.
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