Co-occurrence of genes encoding carbapenemase, ESBL, pAmpC and non-ß-Lactam resistance among Klebsiella pneumonia and E. coli clinical isolates in Tunisia.

This study aimed to investigate the molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae and E. coli isolates obtained from hospitalized patients in Carthagene International Hospital of Tunis. A total of 25 K. pneumoniae and 2 E. coli clinical isolates with reduced susceptibility to carbapenems were recovered. Susceptibility testing and phenotypic screening tests were carried out. ESBL, AmpC, carbapenemase and other antibiotic resistance genes were sought by PCR-sequencing. The presence of plasmid-mediated colistin resistance genes (mcr-1-8) was examined by PCR and the nucleotide sequence of the mgrB gene was determined. The analysis of plasmid content was performed by PCR-Based Replicon Typing (PBRT). The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). All of the isolates produced carbapenemase activity. They showed a great variation in the distribution of ESBL, AmpC, carbapenemase and other plasmid-mediated resistance determinants. K. pneumoniae isolates carried blaNDM-1 (n = 11), blaOXA-48 (n = 11), blaNDM-1 + blaOXA-48 (n = 1), blaNDM-1 + blaVIM-1 (n = 1), blaOXA-204 (n = 1), along with blaCTX-M , blaOXA , blaTEM , blaCMY , blaDHA and blaSHV genes variants on conjugative plasmid of IncL/M, IncR, IncFIIK , IncFIB, and IncHI1 types. Three sequence types ST101, ST307 and ST15 were identified. The mgrB alteration g109a (G37S) was detected in a single colistin-resistant, NDM-1 and OXA-48-coproducing K. pneumoniae isolate. The two E. coli isolates belonged to ST95, co-produced NDM-1 and CTX-M-15, and harboured plasmid of IncFII and IncFIB types. To our knowledge, this is the first report in Tunisia of NDM-1, OXA-48, and CTX-M-15 coexistence in colistin-resistant K. pneumoniae ST15.

Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like ß-Lactamase.

OXA-535 is a chromosome-encoded carbapenemase of Shewanella bicestrii JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most ß-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535. OXA-535 and OXA-436, a plasmid-encoded variant of OXA-535 differing by three amino acids, form a novel cluster of distantly related OXA-48-like carbapenemases. Comparison of blaOXA-535 and blaOXA-436 genetic environments suggests that an ISCR1 may be responsible for blaOXA-436 gene mobilization from the chromosome of Shewanella spp. to plasmids.

Neonatal infections with multidrug-resistant ESBL-producing E. cloacae and K. pneumoniae in Neonatal Units of two different Hospitals in Antananarivo, Madagascar.

BACKGROUND: We investigated the molecular mechanism of ß-lactam resistance in extended-spectrum ß-lactamase (ESBL)-producing Enterobacterial strains isolated in neonatal units of different hospitals in Anatnanarivo, Madagascar. METHODS: Bacteria were identified by standard biochemical methods, disc diffusion antibiograms and Etest. Resistance genes were sought by PCR. Strains were characterized by Rep-PCR (Diversilab), plasmid analysis and rep-typing. RESULTS: From April 2012 to March 2013, 29 ESBL-producing E. cloacae and 15 K. pneumoniae were isolated from blood culture (n = 32) or gastric samples (n = 12) performed at day 0 or 2 from 39/303 newborns suspected of early neonatal infection. These infants were treated with expanded spectrum cephalosporins, due to lack of carbapenems, leading to a high mortality rate (45 %). Isolates recovered were all, but 4, multidrug resistant, particularly to fluoroquinolones (FQ) except for 21 E. cloacae isolates. Isolates produced TEM-1 and CTX-M-15 ß-lactamases and their genes were located on several self-transferable plasmids of variable sizes sizes that could not be linked to a major plasmid incompatibility group. E. cloacae isolates belonged to 6 Rep-types among which two counted for 11 isolates each. The FQ resistant E. cloacae isolates belonged to one clone, whereas the FQ susceptible E. cloacae isolates belonged to four clones. The K. pneumoniae isolates belonged to 9 Rep-types among which one included five isolates. CONCLUSION: This study is the first molecular characterization of ESBL-producing isolates from neonatology units in Madagascar, a country with limited epidemiological data. It revealed an important multi-clonal dissemination of CTX-M-15-producing isolates reflecting both the high community carriage and the very early nosocomial contamination of the neonates.

OXY-2-15, a novel variant showing increased ceftazidime hydrolytic activity.

OBJECTIVES: Klebsiella oxytoca is a member of the family of Enterobacteriaceae and often contains the ß-lactamase blaOXY gene. Although this ß-lactamase does not naturally hydrolyse ceftazidime, this study describes possible in vivo selection of a clinical K. oxytoca isolate showing increased MICs of ceftazidime. METHODS: To reveal the molecular mechanism underlying this unusual resistance phenotype, WGS, cloning, overexpression, MIC and steady-state kinetic studies were performed. RESULTS: A patient was treated for a septic episode with ceftazidime (4 g/day). This therapy was based on earlier culture results in which, amongst others, a K. oxytoca (Velp-1) isolate was identified. After 11 days of treatment, K. oxytoca Velp-2 was isolated from a pus sample drained from the wound. The isolate showed increased resistance to ceftazidime (MIC ≥64 mg/L) compared with the original K. oxytoca isolate (Velp-1). WGS revealed the presence of a novel blaOXY-2 allele, designated blaOXY-2-15, with a two amino acid deletion at Ambler positions 168 and 169 compared with OXY-2-2. Cloning blaOXY-2-15 into Escherichia coli TOP10 resulted in increased MICs of ceftazidime, but reduced MICs of most other ß-lactams compared with OXY-2-2. Steady-state kinetics confirmed the results of the MIC data, showing clearly significant ceftazidime hydrolysis. CONCLUSIONS: This report shows the risk of in vivo selection of ceftazidime-resistant K. oxytoca isolates after prolonged ceftazidime treatment. Furthermore, it is the first known report of a K. oxytoca isolate conferring resistance to ceftazidime by a two amino acid deletion in the omega loop of OXY-2-2.

Many human L1 elements are capable of retrotransposition.

Using a selective screening strategy to enrich for active L1 elements, we isolated 13 full-length elements from a human genomic library. We tested these and two previously-isolated L1s (L1.3 and L1.4) for reverse transcriptase (RT) activity and the ability to retrotranspose in HeLa cells. Of the 13 newly-isolated L1s, eight had RT activity and three were able to retrotranspose. L1.3 and L1.4 possessed RT activity and retrotransposed at remarkably high frequencies. These studies bring the number of characterized active human L1 elements to seven. Based on these and other data, we estimate that 30-60 active L1 elements reside in the average diploid genome.

Harmonisation of in-silico next-generation sequencing based methods for diagnostics and surveillance.

Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436 Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype-phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.

Evaluation of a DNA microarray, the check-points ESBL/KPC array, for rapid detection of TEM, SHV, and CTX-M extended-spectrum beta-lactamases and KPC carbapenemases.

Extended-spectrum beta-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbepenemases) have rapidly emerged worldwide and require rapid identification. The Check-Points ESBL/KPC array, a new commercial system based on genetic profiling for the direct identification of ESBL producers (SHV, TEM, and CTX-M) and of KPC producers, was evaluated. Well-characterized Gram-negative rods (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) expressing various ss-lactamases (KPC-2, SHV, TEM, and CTX-M types) were used as well as wild-type reference strains and isolates harboring ss-lactamase genes not detected by the assay. In addition, phenotypically confirmed ESBL producers isolated in clinical samples over a 3-month period at the Bicetre hospital were analyzed using the Check-Points ESBL/KPC array and by standard PCR. The Check-Points ESBL/KPC array allowed fast detection of all TEM, SHV, and CTX-M ESBL genes and of the KPC-2 gene. The assay allowed easy differentiation between non-ESBL TEM and SHV and their ESBL derivatives. None of the other tested ss-lactamase genes were detected, underlining its high specificity. The technique is suited for Enterobacteriaceae but also for P. aeruginosa and A. baumannii. However, for nonfermenters, especially P. aeruginosa, a 1:10 dilution of the total DNA was necessary to detect KPC-2 and SHV-2a genes reliably. The Check-Points ESBL/KPC array is a powerful high-throughput tool for rapid identification of ESBLs and KPC producers in cultures. It provided definitive results within the same working day, allowing rapid implementation of isolation measures and appropriate antibiotic treatment. It showed an interesting potential for routine laboratory testing.

Intrafamilial transmission of extended-spectrum-beta-lactamase-producing Escherichia coli and Salmonella enterica Babelsberg among the families of internationally adopted children.

OBJECTIVES: International adoption from developing countries has become an increasing phenomenon in recent years. Given the high prevalence of multidrug-resistant (MDR) bacteria in these countries, the adopted children represent a group at risk for both carriage and infection with MDR bacteria. The dynamics of intrafamilial transmission of MDR bacteria after adoption was studied in a prospective study from January 2002 to January 2005. METHODS: Stool samples, taken at the first visit to the outpatient adoption practice and subsequently every month, from the adopted children of an orphanage of Bamako (Mali) and from all the members of their adoptive families were screened for MDR bacteria and bacterial pathogens. Bacteria were characterized by standard biochemical methods, disc diffusion antibiograms, PFGE and plasmid analysis. beta-Lactamase genes were sought by PCR. RESULTS: Over the study period, 52 ESBL-producing Enterobacteriaceae (E-ESBL), with Escherichia coli (56%) being the most prevalent, were isolated from 24/25 adoptees at arrival in France. During follow-up, the transmission of ESBL-producing E. coli and Salmonella enterica Babelsberg between the adoptees and their adoptive family members has clearly been demonstrated for 5/22 families (23%). The mean duration of the carriage for the adopted children was 9 months (1-15 months). CTX-M-15 was the most prevalent resistance gene among the E-ESBLs (93%), while SHV-12 was found among the S. enterica Babelsberg studied. CONCLUSIONS: International travellers, transfer of patients and now adoption may contribute to the global emergence of MDR bacteria. Thus, in addition to the usual screening of adopted children for infectious diseases, additional screening for MDR bacteria should be recommended, at least for children coming from countries with a high prevalence of MDR bacteria.

[KPC carbapenemases: what is at stake in clinical microbiology?]. / Carbapénèmases de type KPC: quel enjeu en microbiologie clinique?

Emergence and dissemination of carbapenem resistance in the world represent a significant threat for management of hospital-acquired infections. From the early 2000s, Enterobacteriaceae that produce Klebsiella pneumoniae carbapenemases (KPC) have initially been reported from the USA and now worldwide, becoming the most important carbapenemase. These KPC producing-bacteria are mostly involved in nosocomial and systemic infections. They are mostly Enterobacteriaceae and more rarely Pseudomonas aeruginosa. KPC beta-lactamases confer decreased susceptibility or resistance to virtually all beta-lactams. Therefore, carbapenems (imipenem, meropenem, ertapenem) may become inefficient for treating enterobacterial infections with KPC-producing bacteria, which are in addition resistant to many other non beta-lactam molecules, leaving only few available therapeutic options. Detection of KPC-producing bacteria may be difficult based on routine antibiotic susceptibility testing. Several phenotypic tests have been proposed, but until now, only molecular methods are reliable techniques for their identification. It is therefore critical to implement efficient infection control measures to detect patients who are colonized or infected with these pathogens in order to limit their spread.

Emerging extensively drug-resistant bacteria (eXDR) in France in 2018.

OBJECTIVE: This 2018 report of Healthcare-Associated Infections Early Warning and Response System (HAI-EWRS) notifications of carbapenemase-producing Enterobacteriaceae (CPE) or glycopeptide-resistant Enterococcus faecium (GRE), and of strains analysed by the National Reference Center for anti-microbial resistance (NRC) aimed to describe the epidemiology of emerging extensively drug-resistant bacteria (eXDR) in France and control measures implemented in hospital settings. PATIENTS AND METHODS: All HAI-EWRS notifications of eXDR received at the national level and all eXDR strains received at the NRC between January 1, 2018 and January 31, 2018 were analysed. Variables analysed were number of cases, number of strains, resistance mechanism, sample type, link with a foreign country, and control measures implemented. RESULTS: In 2018, 1704 CPE notifications and 315 GRE notifications were reported in France, with an increasing trend since 2012 (×6 for CPE, ×3 for GRE), from respectively 364 and 155 hospitals (+66% for CPE, +57% for GRE since 2012). eXDR strains were mainly isolated from rectal screening swabs. Notifications with patients receiving standard precautions were more often associated with outbreaks than notifications with patients receiving contact precautions at admission. NRC received 2674 CPE strains and 775 GRE strains in 2018 (×8.3 and ×2.8 compared with 2012). CONCLUSION: The increasing annual number of eXDR notifications and eXDR strains received by the NRC is multifactorial but reflects a worrying spread of eXDR in France. The number of infections remains low, but this article shows that existing recommendations are not fully implemented.

Two silent substitutions in the PDHA1 gene cause exon 5 skipping by disruption of a putative exonic splicing enhancer.

BACKGROUND: Synonymous mutations within exons may cause aberrant splicing by disrupting exonic splicing enhancer (ESE) motifs in the vicinity of non consensus splice sites. Mutational analysis of PDHA1 revealed only one silent single nucleotide substitution in exon 5 in two unrelated boys and a girl (c.483C>T and c.498C>T variants, respectively). For both patients, pyruvate dehydrogenase complex activity was low and the immunoreactive E1alpha protein was defective in cultured fibroblasts. METHODS AND RESULTS: One of the boys was a somatic mosaic for the c.483C>T variant, as shown by the variable ratio of mutant to normal alleles in fibroblast, lymphocyte and single hair root DNA. Transcript analysis in fibroblasts from the three patients revealed the presence of both normal and truncated cDNAs, with the splicing out of exon 5 predicted to result in a frame shift and premature termination (p.Arg141AlafsX11). The treatment of fibroblasts with emetine before harvesting to prevent nonsense mRNA-mediated decay increased the amount of mutant mRNA. In silico analysis revealed that each variant disrupted a putative SRp55 binding site and that the intron 5 donor splice site (5'ss) contained a weak splicing signal. Transient transfection of COS-7 or Hela cells with hybrid minigene constructs containing wild-type or mutant PDHA1 exon 5, followed by RT-PCR demonstrated that each variant resulted in the incomplete inclusion of PDHA1 exon 5, and that this defect was corrected following the restoration of a perfect consensus sequence for the 5' splice site by site-directed mutagenesis. CONCLUSION: These two synonymous mutations expand the spectrum of rare PDHA1 splicing mutations, all of which are located in non canonical splice sites.

Minor extended-spectrum beta-lactamases.

Extended-spectrum beta-lactamases (ESBLs) are usually plasmid-mediated enzymes that confer resistance to a broad range of beta-lactams. Initially, resistance to third-generation cephalosporins in Gram-negative rods was mainly due to the dissemination of TEM- and SHV-type ESBLs, which are point mutants of the classic TEM and SHV enzymes with extended substrate specificity. During the last ten years, CTX-M-type ESBLs have become increasingly predominant, but less frequent class A beta-lactamases have also been described, including SFO, BES, BEL, TLA, GES, PER and VEB types. While several of these latter are rarely identified, or are very localised, others are becoming locally prevalent, or are increasingly isolated worldwide. In addition, mutations can extend the spectrum of some OXA-type beta-lactamases to include expanded-spectrum cephalosporins, and several of these enzymes are considered to be ESBLs.

Genetic support of extended-spectrum beta-lactamases.

Genes encoding extended-spectrum beta-lactamases (ESBLs) have been reported in a variety of Gram-negative species, mostly in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. They are mostly either TEM or SHV derivatives, CTX-M-like enzymes--now emerging worldwide--or, less frequently, VEB, GES, and PER ESBLs. The mechanisms responsible for their acquisition are very diverse, and mostly are related to insertion sequences (ISs), transposons, class 1 integrons, and also sul1-type integrons containing the ISCR1 element. This diversity of genetic vehicles at the origin of these mobilisation/acquisition processes enhances the spread of ESBLs.

The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST Subcommittee.

Whole genome sequencing (WGS) offers the potential to predict antimicrobial susceptibility from a single assay. The European Committee on Antimicrobial Susceptibility Testing established a subcommittee to review the current development status of WGS for bacterial antimicrobial susceptibility testing (AST). The published evidence for using WGS as a tool to infer antimicrobial susceptibility accurately is currently either poor or non-existent and the evidence / knowledge base requires significant expansion. The primary comparators for assessing genotypic-phenotypic concordance from WGS data should be changed to epidemiological cut-off values in order to improve differentiation of wild-type from non-wild-type isolates (harbouring an acquired resistance). Clinical breakpoints should be a secondary comparator. This assessment will reveal whether genetic predictions could also be used to guide clinical decision making. Internationally agreed principles and quality control (QC) metrics will facilitate early harmonization of analytical approaches and interpretive criteria for WGS-based predictive AST. Only data sets that pass agreed QC metrics should be used in AST predictions. Minimum performance standards should exist and comparative accuracies across different WGS laboratories and processes should be measured. To facilitate comparisons, a single public database of all known resistance loci should be established, regularly updated and strictly curated using minimum standards for the inclusion of resistance loci. For most bacterial species the major limitations to widespread adoption for WGS-based AST in clinical laboratories remain the current high-cost and limited speed of inferring antimicrobial susceptibility from WGS data as well as the dependency on previous culture because analysis directly on specimens remains challenging. For most bacterial species there is currently insufficient evidence to support the use of WGS-inferred AST to guide clinical decision making. WGS-AST should be a funding priority if it is to become a rival to phenotypic AST. This report will be updated as the available evidence increases.

Pyrosequencing for rapid identification of carbapenem-hydrolysing OXA-type beta-lactamases in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii isolates producing carbapenem-hydrolysing oxacillinases are emerging worldwide. These enzymes are divided into four phylogenetic subgroups: OXA-23-like, OXA-51-like, OXA-24-like and OXA-58-like. A PCR-based approach linked to pyrosequencing analysis was developed to identify the genes for these beta-lactamases. Carbapenem-hydrolysing oxacillinases were rapidly and unambiguously identified in a collection of carbapenem-resistant clinical isolates of A. baumannii and Acinetobacter junii. Pyrosequencing may provide a rapid tool for identification of OXA variants, thus avoiding delays inherent in classical sequencing methods.

GES-11-producing Acinetobacter baumannii clinical isolates from Tunisian hospitals: Long-term dissemination of GES-type carbapenemases in North Africa.

Acinetobacter baumannii is an emerging threat in healthcare facilities owing to its ability to be multidrug-resistant (MDR) and to be involved in outbreaks. GES-type extended-spectrum ß-lactamases (ESBLs) have been increasingly identified in A. baumannii. In this study, clinical A. baumannii isolates were characterised using standard biochemical methods and antibiotic susceptibility testing. Antibiotic resistance genes were sought by PCR and sequencing. Genetic support was characterised using S1 nuclease pulsed-field gel electrophoresis (PFGE) mapping, conjugation and electroporation assays. The genetic environment was investigated by PCR, and genetic relatedness was investigated by PFGE. Two MDR A. baumannii clinical isolates susceptible only to colistin and rifampicin were isolated from a tracheal aspirate of a 49-year-old woman hospitalised in 2006 at the Military Hospital of Tunis, Tunisia, and from a tracheal aspirate of a 53-year-old man hospitalised in 2010 at the Institut Orthopédique Mohamed El Kassab of Tunis, Tunisia. PCR revealed that the two isolates harboured the acquired carbapenemase blaOXA-23 and ESBL blaGES-11 genes along with chromosomally-encoded blaOXA-51 and blaADC-like genes. PFGE revealed that these A. baumannii isolates were unrelated; nevertheless, plasmid analysis revealed a similar sized plasmid following electrophoresis of the isolates. In addition, A. baumannii CIP70.10 transformants displayed similar resistance patterns. blaGES-11 was integron-borne and the ISAbaI element was identified upstream of blaOXA-23 and blaADC-like. Here we described two unrelated clinical A. baumannii isolates producing GES-11 ESBL and OXA-23 carbapenemase from two Tunisian hospitals. This work further illustrates the emergence of GES-type ß-lactamases in A. baumannii in North Africa as early as 2006.

Escherichia coli: an old friend with new tidings.

Escherichia coli is one of the most-studied microorganisms worldwide but its characteristics are continually changing. Extraintestinal E. coli infections, such as urinary tract infections and neonatal sepsis, represent a huge public health problem. They are caused mainly by specialized extraintestinal pathogenic E. coli (ExPEC) strains that can innocuously colonize human hosts but can also cause disease upon entering a normally sterile body site. The virulence capability of such strains is determined by a combination of distinctive accessory traits, called virulence factors, in conjunction with their distinctive phylogenetic background. It is conceivable that by developing interventions against the most successful ExPEC lineages or their key virulence/colonization factors the associated burden of disease and health care costs could foreseeably be reduced in the future. On the other hand, one important problem worldwide is the increase of antimicrobial resistance shown by bacteria. As underscored in the last WHO global report, within a wide range of infectious agents including E. coli, antimicrobial resistance has reached an extremely worrisome situation that 'threatens the achievements of modern medicine'. In the present review, an update of the knowledge about the pathogenicity, antimicrobial resistance and clinical aspects of this 'old friend' was presented.

Molecular characterisation of In51, a class 1 integron containing a novel aminoglycoside adenylyltransferase gene cassette, aadA6, in Pseudomonas aeruginosa.

Polymerase chain reaction-amplification and subsequent sequencing of the variable region of a novel integron, In51, from Pseudomonas aeruginosa revealed the presence of a novel aminoglycoside adenylyltransferase gene, aadA6, together with an open reading frame of unknown function, orfD. AADA6 enzyme has only 75% amino acid identity with AADA1 and is able to confer high level resistance to streptomycin and spectinomycin in Escherichia coli.

Role of ser-237 in the substrate specificity of the carbapenem-hydrolyzing class A beta-lactamase Sme-1.

The role of the serine residue found at position 237 in the carbapenemase Sme-1 has been investigated by constructing a mutant in which Ser-237 was replaced by an alanine. The S237A mutant showed a catalytic behavior against penicillins and aztreonam very similar to that of Sme-1. By contrast, S237A was characterized by a reduced catalytic efficiency against cephems, such as cephalothin and cephaloridine. In addition, the weak activity of Sme-1 against the cephamycin cefoxitin was hardly detectable with the mutant enzyme. Finally, the Ser-237-->Ala mutation resulted in a marked decrease in catalytic activity against imipenem, showing that Ser-237 contributes to the carbapenemase activity of the class A beta-lactamase Sme-1.

Insertion sequence-related genetic variation in resting Escherichia coli K-12.

Bacterial subclones recovered from an old stab culture of Escherichia coli K-12 revealed a high degree of genetic diversity, which occurred in spite of a very reduced rate of propagation during storage. This conclusion is based on a pronounced restriction fragment length polymorphism (RFLP) detected upon hybridization with internal fragments of eight resident insertion sequences (IS). Genetic diversity was dependent on the IS considered and, in many cases, a clear consequence of IS transposition. IS5 was particularly active in the generation of variation. All subclones in which IS30 had been active testify to a burst of IS30 transposition. This was correlated with a loss of prototrophy and a reduced growth on rich media. A pedigree of the entire clone could be drawn from the RFLP patterns of the subclones. Out of 118 subclones analyzed, 68 different patterns were found but the putative ancestral population had disappeared. A few patterns were each represented by several subclones displaying improved fitness. These results offer insights into the role of IS elements in the plasticity of the E. coli genome, and they further document that enzyme-mediated DNA rearrangements do occur in resting bacterial cultures.