Departments of 1 Biological Sciences, Box 70703 and 2 Internal Medicine, Box 70622, East Tennessee State University, Johnson City, TN 37614; 3 James H. Quillen Veterans Affairs Medical Center (11C), Mountain Home, TN 37684, USA
Received 18 July 2003; returned 9 October 2003; revised 7 November 2003; accepted 10 November 2003
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Abstract |
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Methods: A rapid, one-step PCR assay conducted on 354 isolates spanning 19841994 distinguished bro alleles in over 97% of the ß-lactamase-producing isolates. Probes of dot blots were used to distinguish PCR failure from non-ß-lactamase-mediated penicillin resistance.
Results: BRO-2 isolates comprised 010% of the population per year with no evidence of a decline over time. All ß-lactamase producers exceeded the clinical threshold for penicillin resistance. Bimodality of penicillin MICs for ß-lactamase producers was caused by variation within BRO-1 rather than differences between BRO-1 and BRO-2. Non-ß-lactamase factors also confer resistance to penicillin and may contribute to the BRO-1 bimodality. The 13 BRO-2 isolates were associated with diverse genotypes within which there was evidence of epidemiologically linked clusters. The exclusive association of BRO-2 with four unrelated genotypes suggested maintenance of BRO-2 by recurrent mutation or horizontal exchange.
Conclusions: The relative rarity of BRO-2 throughout the study, the absence of a declining temporal trend, and genetic diversity within BRO-2 all failed to support the hypothesis that BRO-2 was more common in the past and has been selectively replaced by BRO-1.
Keywords: disease transmission, molecular epidemiology, selection
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Introduction |
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The nucleotide sequence of the bro-2 allele differs from bro-1 by five nucleotides within the coding region, one of which causes an amino acid replacement of unknown significance. The upstream region of bro-1 includes 21 bp encompassing a second copy of a 16 bp repeat motif that is absent in bro-2 and is presumed to function as a promoter enhancer.2
BRO-2 is relatively rare, invariably occurring in less than 15% of isolates.1,3 On the basis of sequence similarity between bro-2 and bro-negative isolates, Bootsma et al.4 hypothesized that a bro-2-like allele was originally transferred into M. catarrhalis and that bro-1 was generated by a duplication in the promoter with subsequent spread enhanced by selection for more active enzymic activity via greater enzyme production.
This study examined evidence for the bro-1 replacement hypothesis, investigated the relationship between allelic types and antibiotic resistance phenotypes, documented the role of non-ß-lactamase factors in penicillin resistance and tested the efficacy of a one-step, length-based PCR assay to distinguish bro alleles.
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Materials and methods |
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Assays were conducted on 354 isolates composed of 2550 randomly selected isolates from each of the 10 years (July 1984 to June 1994) represented in a collection of M. catarrhalis from the Veterans Affairs Medical Center at Mountain Home, TN (VAMC). Details of the collection and methods for isolate selection, antibiotic testing, DNA extraction, and genotyping are given in Walker & Levy5 and Walker et al.6
ß-Lactamase gene assays
PCR primers amplified nucleotides 216 to +19 of the bla locus and encompassed the 21 bp difference that distinguishes the two bro alleles (GenBank U49269 and Z54180). Primer sequences were left: 5'-CACCCYGTRGGACAAGC-3' and right: 5'-AATGACGGCGTTGCATC-3'. PCR products of 235 bp (bro-1) or 214 bp (bro-2) were distinguished on 2.0% agarose gels. Isolates that failed to generate a bro allele PCR product were tested for the presence of the bla gene using PCR primers encompassing 862 bp of the 945 bp of the coding region. Primer sequences were left: 5'-TTTGGATTGGGGTGAATGAT-3' and right: 5'-TGGGGCTGGGTGATAAATAG-3'. PCR protocols consisted of 94°C for 10 min followed by 25 cycles of: 94°C for 30 s, 55°C for 60 s, 72°C for 40 s and ending with 72°C for 7 min. ATCC 43627, 43628 and 25238 were used as BRO-1, BRO-2 and BRO-negative controls, respectively. All negative PCRs were tested twice and DNAs were demonstrated to be suitable PCR substrates in control assays. Digoxigenin-labelled probes for dot blots were constructed by PCR using DNA from ATCC 43627 and BRO-1 clinical isolate 327.
Statistical analysis
Penicillin MICs for BRO-1 and BRO-2 samples were compared using ANOVA.
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Results |
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Discussion |
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Two observations from this study fail to support the hypothesis that BRO-2 was more common in the past and its frequency declined as a result of replacement by BRO-1. First, BRO-2 was absent in isolates from the early collection years, a period when ß-lactamase producers were less common,6 and second, when present, BRO-2 showed a sporadic, rather than declining temporal distribution. Incomplete collections render our analysis and data from other studies inadequate for detecting processes that preceded the early 1980s. Nevertheless, the presence of BRO-2 in diverse, but often rare phylogenetic lineages, combined with prior evidence that the transition to penicillin resistance was not mediated by population bottlenecks or a selective sweep,5 suggests generation of bro-2 alleles by either recurrent mutation or spread via horizontal transmission.
Phylogenetic and epidemiological associations among BRO-2 isolates suggest minor fluctuations in frequency reflect brief bouts of clonal expansion rather than long-term trends caused by selection. For example, temporal clusters of genetically identical BRO-2 isolates accounted for over half (8/13) of the BRO-2 isolates and these appeared to have arisen via patient-to-patient transmission. The frequency of BRO-2 isolates is probably an equilibrium balancing increases by mutation and clonal expansion with decreases caused by either genetic drift or selection. If BRO-1 is under positive selection then the sporadic and continued occurrence of BRO-2 may be explained by recurrent mutation and horizontal exchange, both of which are consistent with observations of BRO-2 in unrelated genotypes as shown in our study and in Bootsma et al.7
Trimodality in the penicillin-susceptibility spectrum
Trimodal patterns of penicillin MICs uncovered in earlier studies led to the hypothesis that the three peaks corresponded to BRO-negative, BRO-2 and BRO-1 strains.6,8,9 The current survey has shown that that hypothesis was not correct but rather, the trimodality is caused by a unimodal pattern in BRO-negative isolates with a peak in the susceptible range (<0.03 mg/L) and bimodality within BRO-1 isolates with peaks at 1 and >16 mg/L (Table 2).
Factors other than the characterized differences between BRO-1 and BRO-2 must underlie the BRO-1 bimodal pattern. Similar hypotheses of additional factors have been offered to explain the range of ampicillin MICs among ß-lactamase producers10 and the overlap in ampicillin MICs among BRO types.3
Non-ß-lactamase-mediated resistance factors as MIC modifiers
Twelve ß-lactamase-negative isolates, representing five different genotypes, displayed MICs with either reduced susceptibility or clinically-relevant penicillin resistance (0.25 mg/L). These strains tested negative for the presence of the bla gene by two independent PCR assays and a genomic dot blot assay, and they failed to hydrolyse nitrocefin. Penicillin MICs for these isolates ranged from 0.06 to 0.25 mg/L, conclusively demonstrating that non-ß-lactamase resistance factors can confer clinical resistance in M. catarrhalis.
In Neisseria gonorrhoeae, non-ß-lactamase-mediated penicillin resistance developed through the gradual accumulation of mutations, each leading to a minor reduction in susceptibility. These mutations gave rise to an altered penicillin-binding protein (PBP), enhanced efflux pumps, and decreased membrane permeability.1113 Altered PBPs confer resistance specifically to ß-lactam agents, but altered efflux and permeability confer reduced susceptibility to non-ß-lactam agents including macrolides.12,13 Similar non-ß-lactamase resistance mechanisms may underlie prior observations of antibiotic-specific changes in susceptibility in M. catarrhalis at the VAMC. For example, cefamandole resistance declined after the drug was no longer used at the medical centre, although resistance to penicillin and the proportion of ß-lactamase producers continued to rise in the population.6 Additionally, a significant temporal population trend toward increasing clarithromycin MICs was restricted to the ß-lactamase producers,6 a phenotype consistent with determinants conferring altered efflux or permeability.
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Acknowledgements |
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Footnotes |
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References |
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2 . Bootsma, H. J., van Dijk, H., Vorhoef, J. et al. (1996). Molecular characterization of the BRO ß-lactamase of Moraxella (Branhamella) catarrhalis. Antimicrobial Agents and Chemotherapy 40, 96672.[Abstract]
3 . Fung, C. P., Yeo, S. F. & Livermore, D. M. (1994). Susceptibility of Moraxella catarrhalis isolates to ß-lactam antibiotics in relation to ß-lactamase pattern. Journal of Antimicrobial Chemotherapy 33, 21522.[Abstract]
4 . Bootsma, H. J., van Dijk, H., Vauterin, P. et al. (2000). Genesis of BRO ß-lactamase-producing Moraxella catarrhalis: evidence for transformation-mediated horizontal transfer. Molecular Microbiology 36, 93104.[CrossRef][ISI][Medline]
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13 . Hagman, K. E., Pan, W., Spratt, B. G. et al. (1995). Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system. Microbiology 141, 61122.[Abstract]
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