Bernd Wiedemann

Evolution of plasmid-coded resistance to broad-spectrum cephalosporins.

A clinical isolate of Klebsiella ozaenae with transferable resistance to broad-spectrum cephalosporins produces a beta-lactamase determined by plasmid pBP60. The beta-lactamase had the same isoelectric point as SHV-1 (7.6). From heteroduplex analysis, an extensive homology between the two bla genes could be deduced; therefore, the new beta-lactamase was designated SHV-2. Enzymatic studies revealed that SHV-2 was able to hydrolyze broad-spectrum cephalosporins due to an increased affinity of these compounds for the enzyme. The assumption that SHV-2 is a natural mutant of SHV-1 was strongly supported by the isolation of a laboratory mutant of SHV-1 that showed activities similar to those of SHV-2. Images

Resistance Mechanisms of Multiresistant Pseudomonas aeruginosa Strains from Germany and Correlation with Hypermutation

ABSTRACT In this study, we analyzed the mechanisms of multiresistance for 22 clinical multiresistant and clonally different Pseudomonas aeruginosa strains from Germany. Twelve and 10 strains originated from cystic fibrosis (CF) and non-CF patients, respectively. Overproduction of the efflux systems MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM was studied. Furthermore, loss of OprD, alterations in type II topoisomerases, AmpC overproduction, and the presence of 25 acquired resistance determinants were investigated. The presence of a hypermutation phenotype was also taken into account. Besides modifications in GyrA (91%), the most frequent mechanisms of resistance were MexXY-OprM overproduction (82%), OprD loss (82%), and AmpC overproduction (73%). Clear differences between strains from CF and non-CF patients were found: numerous genes coding for aminoglycoside-modifying enzymes and located, partially in combination with β-lactamase genes, in class 1 integrons were found only in strains from non-CF patients. Furthermore, multiple modifications in type II topoisomerases conferring high quinolone resistance levels and overexpression of MexAB-OprM were exclusively detected in multiresistant strains from non-CF patients. Correlations of the detected phenotypes and resistance mechanisms revealed a great impact of efflux pump overproduction on multiresistance in P. aeruginosa. Confirming previous studies, we found that additional, unknown chromosomally mediated resistance mechanisms remain to be determined. In our study, 11 out of 12 strains and 3 out of 10 strains from CF patients and non-CF patients, respectively, were hypermutable. This extremely high proportion of mutator strains should be taken into consideration for the treatment of multiresistant P. aeruginosa.

In Vitro Activities of Ertapenem (MK-0826) against Recent Clinical Bacteria Collected in Europe and Australia

ABSTRACT Ertapenem (MK-0826, L-749,345) is a 1-β-methyl carbapenem with a long serum half-life. Its in vitro activity was determined by broth microdilution against 3,478 bacteria from 12 centers in Europe and Australia, with imipenem, cefepime, ceftriaxone, and piperacillin-tazobactam used as comparators. Ertapenem was the most active agent tested against members of the familyEnterobacteriaceae, with MICs at which 90% of isolates are inhibited (MIC90s) of ≤1 μg/ml for all species. Ertapenem also was more active than imipenem against fastidious gram-negative bacteria and Moraxella spp.; on the other hand, ertapenem was slightly less active than imipenem against streptococci, methicillin-susceptible staphylococci, and anaerobes, but its MIC90s for these groups remained ≤0.5 μg/ml.Acinetobacter spp. and Pseudomonas aeruginosawere also much less susceptible to ertapenem than imipenem, and mostEnterococcus faecalis strains were resistant. Ertapenem resistance, based on a provisional NCCLS MIC breakpoint of ≥16 μg/ml, was seen in only 3 of 1,611 strains of the familyEnterobacteriaceae tested, all of them Enterobacter aerogenes. Resistance was also seen in 2 of 135 anaerobes, comprising 1 Bacteroides fragilis strain and 1Clostridium difficile strain. Ertapenem breakpoints for streptococci have not been established, but an unofficial susceptibility breakpoint of ≤2 μg/ml was adopted for clinical trials to generate corresponding clinical response data for isolates for which MICs were as high as 2 μg/ml. Of 234 Streptococcus pneumoniae strains tested, 2 required ertapenem MICs of 2 μg/ml and one required an MIC of 4 μg/ml, among 67 non-Streptococcus pyogenes, non-Streptococcus pneumoniae streptococci, single isolates required ertapenem MICs of 2 and 16 μg/ml. These streptococci also had diminished susceptibilities to other β-lactams, including imipenem as well as ertapenem. The Etest and disk diffusion gave susceptibility test results in good agreement with those of the broth microdilution method for ertapenem.

Chromosomal beta-lactamases of Enterobacter cloacae are responsible for resistance to third-generation cephalosporins.

About 70% of all Enterobacter cloacae strains tested possessed one of two species-specific beta-lactamases. These enzymes, E. cloacae beta-lactamase A and E. cloacae beta-lactamase B, with isoelectric points of 8.8 and 7.8, respectively, had the same pH and temperature optima. Both showed similar enzyme kinetics and were inhibited by cloxacillin but not by p-chloromercuribenzoate. E. cloacae beta-lactamase B appeared to be identical with the enzyme of E. cloacae P99. By a mutation in a regulatory gene, inducible enzyme production could be converted into constitutive expression. In E. cloacae, both enzymes did not hydrolyze third-generation cephalosporins, but they were solely responsible for resistance toward these drugs. This was demonstrated by the characterization of Escherichia coli strains expressing an identical resistance pattern after transfer of the corresponding Enterobacter gene. Images

Development of resistance to nalidixic acid and the fluoroquinolones after the introduction of norfloxacin and ofloxacin.

Since 1975, the group Resistenz of the Paul-Ehrlich-Gesellschaft für Chemotherapie has monitored the development of resistance in isolates of members of the family Enterobacteriaceae, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis in the Federal Republic of Germany, West Berlin, Austria, and Switzerland. Despite a marked increase in the use of 4-quinolones, there was no increase in the percentages of nalidixic acid-resistant strains of Enterobacteriaceae between 1975 and 1986. However, different bacterial species showed considerable variation, and there were also considerable differences in the percentages of nalidixic acid-resistant strains of Enterobacteriaceae from different centers. The frequency of resistance to fluoroquinolones was unchanged from 1983 to 1986 and was less than 4% in all species except P. aeruginosa. In this species, there was an increase from about 3 to 10% from 1983 to 1986 for strains for which the MIC was fourfold above the mode MIC of ciprofloxacin, enoxacin, and ofloxacin.

Brief report: Resistance development to fluoroquinolones in Europe

We did not observe an increase in quinolone-resistant strains in recent years despite a dramatic increase in drug usage. P. aeruginosa strains should be carefully monitored in the future since a trend to increased MICs seems obvious. Epidemiologic data on resistance have to be evaluated carefully, and special interest must be focused on the breakpoint in relation to the normal distribution of MICs. Conclusions can be drawn only if the increased numbers of strains are clearly separated from the normal distribution.

Mechanisms of quinolone resistance

SummaryTwo mechanisms of resistance to fluoroquinolones are known: (i) alteration of the molecular target of quinolone action — DNA gyrase, and (ii) reduction of the quinolone accumulation. Mutations altering the N-terminus of the gyrase A subunit, especially those around residues Ser83 and Asp87, significantly reduce the susceptibilities towards all quinolones, while alterations of the gyrase B subunit are rarely found and are of minor importance. Reduced drug accumulation is associated with alterations of the outer membrane protein profile in gram-negative bacteria. Such mutations include the marA locus inEscherichia coli and result in low level resistance towards quinolones and unrelated drugs. Increased activity of naturally existing efflux systems, such as the transmembrane protein NorA of staphylococci, may also lead to reduced accumulation in gram-positive and gram-negative bacteria. Clinical fluoroquinolone resistance is rarely found in intrinsically highly susceptible organisms such asEnterobacteriaceae and involves a combination of at least two mutations. In contrast, species with moderate intrinsic susceptibility such asCampylobacter jejuni, Pseudomonas aeruginosa, andStaphylococcus aureus require only one mutation to become clinically resistant. As a consequence development of resistance during therapy may result from acquisition of already resistant strains in the case of susceptible species, and selection of mutants in the case of less susceptible species.ZusammenfassungZwei Mechanismen, die zur Fluorchinolonresistenz führen, sind bekannt: (I) Veränderung der molekularen Zielstruktur für die Chinolonwirkung — der DNS-Gyrase und (II) Verringerung der Chinolonaufnahme. Mutationen im N-terminalen Bereich der Gyrase A-Untereinheit, insbesondere um Positionen Ser83 und Asp87, reduzieren deutlich die Empfindlichkeit gegenüber allen Chinolonen. Veränderungen der Gyrase B-Untereinheit dagegen sind selten und von untergeordneter Bedeutung. Verringerung der Chinolonaufnahme geht einher mit Veränderungen des Musters der Äußeren Membran-Proteine bei gramnegativen Bakterien. Solche Mutationen betreffen unter anderem denmarA Locus inEscherichia coli und bewirken eine geringgradige Resistenz gegenüber Chinolonen und nicht verwandten Substanzen. Eine gesteigerte Aktivität von natürlicherweise vorkommenden Efflux-Systemen, wie zum Beispiel das Transmembranprotein NorA bei Staphylokokken, können ebenfalls zu einer verringerten Aufnahme sowohl in grampositiven als auch in gramnegativen Bakterien führen. Klinische Resistenz gegenüber Fluorchinolonen findet sich selten in normalerweise hochempfindlichen Organismen wie den Enterobacteriaceen und beruht auf einer Kombination von mindestens zwei Mutationen. Dagegen genügt bei weniger empfindlichen Spezies, wieCampylobacter jejuni, Pseudomonas aeruginosa oderStaphylococcus aureus bereits eine Mutation, um klinische Resistenz zu bewirken. Das bedeutet, daß im Falle der hochempfindlichen Spezies Resistenzentwicklung unter der Therapie auf der Übertragung bereits resistenter Zellen beruht, während bei weniger empfindlichen Spezies Mutanten erst selektiert werden.

Natural antibiotic susceptibilities of Edwardsiella tarda, E. ictaluri, and E. hoshinae

ABSTRACT The natural antibiotic susceptibilities to 71 antibiotics of 102Edwardsiella strains belonging to E. tarda (n = 42), E. ictaluri(n = 41), and E.hoshinae (n = 19) were investigated. MICs were determined using a microdilution procedure according to NCCLS criteria and German standards. All edwardsiellae were naturally sensitive to tetracyclines, aminoglycosides, most β-lactams, quinolones, antifolates, chloramphenicol, nitrofurantoin, and fosfomycin. Edwardsiella species were naturally resistant to macrolides, lincosamides, streptogramins, glycopeptides, rifampin, fusidic acid, and oxacillin. Although slight species-dependent differences in natural susceptibilities to some antibiotics (e.g., macrolides and cefaclor) were seen, differences in natural susceptibility affecting clinical assessment criteria were only seen with benzylpenicillin. Whereas E. tarda was naturally resistant to benzylpenicillin, E. hoshinae was naturally sensitive. Natural sensitivity and resistance to this penicillin were found among the strains of E. ictaluri.The observed oxacillin sensitivity of E. ictaluriwas attributed to the failure of the species to grow at higher salt concentrations found in oxacillin-containing microtiter plates. The present study describes a database concerning the natural susceptibility of Edwardsiella species to a wide range of antibiotics, which can be applied to validate forthcoming antibiotic susceptibility tests of these microorganisms.

Natural antibiotic susceptibility of Providencia stuartii, P. rettgeri, P. alcalifaciens and P. rustigianii strains.

The natural antibiotic susceptibility of 38 Providencia rettgeri, 35 P. stuartii, 23 P. alcalifaciens and 20 P. rustigianii strains was examined. MIC values were determined by a microdilution procedure and evaluated by a table calculation programme. P. stuartii was the least susceptible Providencia sp. and was naturally resistant to tetracyclines, some penicillins, older cephalosporins, sulphamethoxazole and fosfomycin and to antibiotics to which other species of Enterobacteriaceae are also resistant. It was naturally sensitive to modern penicillins and cephalosporins, carbapenems and aztreonam, but its susceptibility to aminoglycosides and quinolones was difficult to assess. P. alcalifaciens and P. rustigianii strains were the most susceptible Providencia spp. They were naturally sensitive or intermediate to tetracyclines and sensitive to aminoglycosides and quinolones. Susceptibility to sparfloxacin, biapenem and sulphamethoxazole permitted the discrimination of P. alcalifaciens and P. rustigianii strains. The natural antibiotic susceptibility of P. rettgeri strains was between that of P. stuartii and that of the other providenciae. P. rettgeri was resistant to tetracyclines and fosfomycin, but more susceptible to aminoglycosides, quinolones, fosfomycin and numerous beta-lactam antibiotics than P. stuartii. A database is described of the natural antibiotic susceptibilities of Providencia spp. It can be used for the validation of antibiotic susceptibility test results of these micro-organisms.

Detection of VIM-2 Metallo-β-Lactamase in Pseudomonas aeruginosa from Germany

The emergence of metallo-β-lactamase (MBL)-producing pathogens is an increasing therapeutic problem. These enzymes have a broad-substrate spectrum; they hydrolyze all β-lactams except for the monobactam aztreonam. At the present time, there is no clinically useful inhibitor available. Four