Lucinda M. C. Hall

Persistence of sulphonamide resistance in Escherichia coli in the UK despite national prescribing restriction

BACKGROUND There is a clear association between heavy antimicrobial consumption within a population and the frequent recovery of resistant bacteria, but whether a reduction in antimicrobial use can reverse this process is less clear. We investigated the effect of a national restriction of sulphonamide prescribing in the UK on the prevalence of sulphonamide resistance in Escherichia coli. METHODS Consecutive clinical isolates of E coli were collected at the Royal London Hospital in 1991 and 1999. These collections, each of more than 350 isolates, were compared. Minimum inhibitory concentrations of sulphamethoxazole and eight other antimicrobials were determined. The presence and locations of sulphonamide-resistance genes were examined by PCR, plasmid extraction, Southern hybridisation, and transconjugation. FINDINGS Despite a huge decrease in sulphonamide prescriptions (from 3,208,000 [corrected] prescriptions per year in 1991 to 77,000 [corrected] in 1999), the frequency of resistance remained high in 1999 (165/359 [46.0%] vs 143/360 [39.7%] in 1991; difference 6.2% [95% CI 20.9 to 13.3]). Integron-borne sulI was present in 16.4% of isolates in 1991 and 17.5% in 1999. The prevalence of sulII increased from 26.7% in 1991 to 36.5% in 1999 (difference 9.8% [3.1 to 16.5] p=0.0046). SulII was located on large plasmids, at least some of which were conjugative multiresistance determinants. INTERPRETATION These results show that a huge decrease in antibiotic prescribing does not necessarily reduce resistance within a useful time. The main reason seems to be the genetic linkage of the index resistance to other resistance determinants.

OXA-11, an extended-spectrum variant of OXA-10 (PSE-2) beta-lactamase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa 455, isolated in Ankara, Turkey, produced a pI 6.2 beta-lactamase determined by plasmid pMLH53 and resisted all beta-lactams except carbapenems. This beta-lactamase, named OXA-14, corresponded to OXA-10 (PSE-2) except that aspartate replaced glycine at position 157 and thus is intermediate between OXA-10 and OXA-11, which has aspartate at position 157 and a further substitution at position 143.

Complexity and Diversity of Klebsiella pneumoniae Strains with Extended-Spectrum β-Lactamases Isolated in 1994 and 1996 at a Teaching Hospital in Durban, South Africa

ABSTRACT β-Lactamase production was investigated in cultures of 25Klebsiella pneumoniae isolates isolated at a hospital in Durban, South Africa, in 1994 and 1996. Twenty of these isolates gave ceftazidime MIC/ceftazidime plus clavulanate MIC ratios of ≥8, implying production of extended-spectrum β-lactamases (ESBLs), and DNA sequencing identified an ESBL gene (blaTEM-53) in a further two isolates. Pulsed-field gel electrophoresis (PFGE) defined 4 distinct strains among the 12 isolates collected in 1994 and 9 distinct strains among the 13 isolates collected in 1996. In three cases, multiple isolates from single patients varied in their PFGE profiles and antibiograms, implying mixed colonization or infection. Isoelectric focusing and DNA hybridization found both TEM and SHV enzymes and their genes in all 25 isolates. Many isolates had multiple identical or different β-lactamase gene variants, with at least 84blaSHV and blaTEM gene copies among the 25 organisms. Sequencing identified the genes for the SHV-1, -2, and -5 enzymes and for four new SHV types (SHV-19, -20, -21, and -22). These new SHV variants had novel mutations remote from sites known to affect catalytic activity. Sequencing also found the genes for TEM-1, TEM-53, and one novel type, TEM-63. All the isolates had multiple and diverse plasmids. These complex and diverse patterns of ESBL production and strain epidemiology are far removed from the concept of an ESBL outbreak and suggest a situation in which ESBL production has become endemic and in which evolution is generating a wide range of enzyme combinations. This complexity and diversity complicates patient management and the design of antibiotic use policies.

A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens

An acetylcholinesterase (AChE) gene, Ace.x, showing 93% identity of deduced amino acid sequence to Anopheles stephensi Ace has been cloned from a Culex pipiens strain homozygous for insensitive AChE (iAChE) mediated insecticide resistance. DNA sequence of genomic DNA clones identified exons 2–5. RFLP of six clones indicated four possible alleles. Linkage analysis located Ace.x to chromosome I, less than 0.8 centimorgans from the sex locus, whereas the locus conferring resistance was 2.0 centimorgans from plum‐eye on chromosome II. Ace.1 coding for AChE1, which is associated with resistance, is therefore autosomal. We propose that Ace.x is the recently postulated Ace.2 coding for the biochemically distinct AChE2, which is not associated with resistance.

Evaluation of Semiautomated Multiplex PCR Assay for Determination of Streptococcus pneumoniae Serotypes and Serogroups

ABSTRACT A semiautomated method for the determination of five serotypes and three serogroups in Streptococcus pneumoniae was developed. Primers specific for serotypes 1, 3, 14, 19F, and 23F and serogroups 6, 19, and 23 were combined in three multiplex PCRs. Products were separated by capillary electrophoresis with a 7-min run time, and a serotype or serogroup was assigned on the basis of fragment size. The method was used to test 93 clinical isolates, and all isolates of the serotypes concerned were correctly detected. The strategy would allow the detection of multiple serotypes in a single sample. Detection of additional serotypes could be included as capsule locus sequences become available.

Distribution and characteristics of Escherichia coli clonal group A

Among 1,102 recent Escherichia coli clinical isolates, clonal group A was identified in 17 of 20 (U.S. and non-U.S.) geographic locales, mainly among U.S. isolates (9% vs. 3%; p < 0.001) and those resistant to trimethoprim-sulfamethoxazole (10% vs. 1.7%; p < 0.001). The extensive antimicrobial resistance and virulence profiles of clonal group A may underlie its recent widespread emergence.

Are point mutations or DNA rearrangements responsible for the restriction fragment length polymorphisms that are used to type bacteria

Restriction fragment length polymorphisms (RFLPs) detected in total DNA or in rRNA genes are widely used to differentiate strains of bacteria. The changes accounting for these polymorphisms and the extent of genomic difference that they reflect are generally unknown. In this report, several methods have been used to examine the DNA differences between nine Enterococcus faecalis isolates. Restriction fragments from total DNA and from rRNA genes were compared between isolates using four and five different restriction enzymes, respectively. The proportion of polymorphic fragments detected was greater with total DNA than with rRNA gene patterns, but depended considerably on the restriction enzyme used. DNA changes underlying nine RFLPs were investigated by using the polymorphic fragments as probes to test for alteration in the position of recognition sites of other enzymes. Two polymorphisms were deduced to result from point mutation in a restriction site. Six were judged to result from DNA rearrangements, five of which involved deletion/insertion of the entire probe fragment. The results demonstrate that DNA rearrangements may be responsible for a high proportion of RFLPs used to differentiate and type strains of bacteria. While this does not limit the utility of such methods, it does preclude calculation of overall DNA sequence conservation from similarities in restriction pattern between isolates. DNA sequence determination of the 16S-23S rRNA intergenic spacer of three isolates revealed minimal base substitutions (less than 1%), suggesting that overall sequence divergence between the isolates may be low.

The acetycholinesterase gene ofAnopheles stephensi

Summary1.The acetylcholinesterase (AChE) gene from the important malaria vectorAnopheles stephensi has been isolated by homology to theDrosophila acetylcholinesterase gene.2.The complete sequence and intron-exon organization has been determined. The encoded protein has 69% identity toDrosophila AChE and 38 and 36% identity toTorpedo AChE and human butyrylcholinesterase, respectively.

Cloning and Sequence Analysis of the 10 kDa Antigen Gene of Mycobacterium tuberculosis

The gene encoding a major protein antigen of Mycobacterium tuberculosis has been cloned and sequenced using oligonucleotide probes derived from the N-terminal sequence of the analogous protein from Mycobacterium bovis BCG. The gene analysis revealed a sequence encoding a protein of 99 amino acid residues, with a molecular mass of 10.7 kDa. Computer prediction suggests that the protein contains three T-cell-determined epitopes (of which one has been demonstrated experimentally) and three B-cell-determined epitopes. The protein sequence was homologous to two prokaryote heat-shock proteins and the gene possessed heat-shock-like promoter sequences upstream of the initiation codon. A hairpin loop identified in the upstream sequence may also be important in regulation of the gene.

Multiple mutations modulate the function of dihydrofolate reductase in trimethoprim-resistant Streptococcus pneumoniae.

ABSTRACT Trimethoprim resistance in Streptococcus pneumoniaecan be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC50], 4.2 μM) than was the DHFR from strain CP1015 (IC50, 0.09 μM). However,Km values indicated a lower affinity for the enzymes natural substrates (Km for dihydrofolate [DHF], 3.1 μM for CP1015 and 27.5 μM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, Kmvalues for DHF were reduced (9.2 to 15.2 μM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC50 of up to 13.7 μM). It is postulated that carriage of the mutation M53-I—which, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR—is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates.