Promiscuous transfer of drug resistance in gram-negative bacteria

Bacterial conjugation is a major mechanism for the spread of antibiotic-resistance genes in pathogenic organisms. In gram-negative bacteria, broad-host-range drug-resistance plasmids mediate genetic exchange between many unrelated species. The mechanism of conjugation encoded by the broad-host-range IncP plasmid RK2 has been studied in detail. The location and sequence of the transfer origin of RK2 has been determined. Several barriers limit plasmid transfer between unrelated bacteria: interactions at the cell surface may prevent effective mating contact, restriction systems may degrade foreign DNA, or the plasmid may not replicate in the new host. RK2 has evolved specific mechanisms by which it overcomes these barriers; this plasmid can mediate the transfer of resistance to most gram-negative bacteria.     

beta-Lactam resistance in gram-negative bacteria: global trends and clinical impact.

Microbial drug resistance is an inescapable consequence of the utilization of antimicrobial agents in a given environment. Nowhere is the importance of resistance more evident than among agents of the beta-lactam family. Trends toward increased resistance can be seen among fastidious gram-negative bacteria like Haemophilus influenzae, where ampicillin resistance varies from 1% to 64% globally. For Escherichia coli, ampicillin resistance has risen to > or = 50% in high-risk populations, and resistance to third-generation cephalosporins is now being seen in certain areas. Inducible beta-lactamases have been responsible for increasing multiple beta-lactam resistance among certain Enterobacteriaceae and Pseudomonas aeruginosa, and this has been associated with increased use of newer cephalosporins. Xanthomonas maltophilia with its two inducible beta-lactamases is becoming an increasingly important nosocomial pathogen, especially in areas of heavy imipenem utilization. Only through the recognition of factors associated with increasing resistance and the mechanisms responsible can strategies be designed for minimizing beta-lactam resistance.     

Antibiotic resistance among gram-negative bacteria of lower respiratory tract secretions in hospitalized patients

Data on antibiotic resistance pattern of gram-negative bacterial isolates of lower respiratory tract secretions of hospitalized patients were fed into WHONET computer and analyzed for the year 1999. Out of 860 samples, 269 (31.2%) were culture positive. Gram-negative bacteria (GNB) accounted for 238 (88.4%) positive samples. Non-fermenting gram-negative bacteria (NFGNB) were found in 34% samples, the other common ones being Klebsiella spp (29.8%) and Pseudomonas spp (17.2%). GNB isolates from tracheal aspirates and sputum were 132 (55.4%) and 106 (44.5%) respectively. Adults (32.7%) and elderly patients (24.3%) recorded higher isolation of GNB as compared to pediatric patients (1.6%). The highest mean resistance among predominant GNB in both tracheal aspirate (96.6%) and sputum (86.9%) was noted to ampicillin while the lowest mean resistance in tracheal aspirate (28%) and sputum (14.3%) was to amikacin. NFGNB of tracheal aspirates and sputum showed highest resistance of 50% and 32% to amikacin, respectively. Pseudomonas spp showed the highest variation in the resistance pattern between tracheal aspirates and sputum samples. Overall mean resistance was highest among tracheal aspirate isolates compared to sputum isolates.     

Sensitivity of gram-negative bacteria to six aminoglycoside antibiotics

Summary An in vitro study of the susceptibility of 201 newly isolated strains of gramnegative bacteria to six aminoglycoside antibiotics (kanamycin, amikacin, gentamicin, tobramycin, sisomicin and netilmicin) was performed by the twofold dilution method in fluid medium. Both the minimal inhibitory concentration and the minimal bacteridical concentration were determined. Overall, tobramycin seemed the most effective of the drugs studied. Netilmicin, the new derivative from sisomicin, compared favourably with the other drugs tested, but may, on theoretical grounds, offer the additional advantage of retained efficacy in the face of developing bacterial resistance. Not unexpectedly, amikacin appeared to be the most promising of the drugs studied in its action against Pseudomonas aeruginosa. Amikacin and netilmicin appeared to be the most effective of this group of antibiotics against Klebsiella species.

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Empfindlichkeit gramnegativer Bakterien gegenüber sechs Aminoglykosid-Antibiotika

Zusammenfassung Eine In-vitro-Studie über die Empfindlichkeit von 201 neu isolierten gramnegativen Bakterienstämmen gegenüber sechs Aminoglykosid-Antibiotika (Kanamycin, Amikacin, Gentamicin, Tobramycin, Sisomicin und Netilmicin) wurde mit Hilfe eines zweifachen Reihenverdünnungstests durchgeführt. Sowohl die minimale Hemmkonzentration als auch die minimale bakterizide Konzentration wurden bestimmt. Insgesamt erwies sich Tobramycin als das wirksamste der geprüften Medikamente. Im Vergleich zu den anderen untersuchten Medikamenten erzielte Netilmicin, das neue Derivat von Sisomicin, günstige Resultate. Außerdem hat dieses Medikament, rein theoretisch gesehen, den Vorteil einer stabileren Empfindlichkeit bei auftretender bakterieller Resistenz. Erwartungsgemäß erwies sich Amikacin als das zuverlässigste aller getesteten Medikamente gegen Pseudomonas aeruginosa. Als wirksamste Antibiotika gegen Klebsiella waren Amikacin und Netilmicin zu bezeichnen.

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Although this article does not deal directly with antibiotic treatment, the article was accepted for publication because resistance to the aminoglycoside antibiotics is attracting increasing attention in antibacterial therapy in hospitals. This subject will be dealt with again in a coming issue of INFECTION in an article byH. Knothe andV. Krcméry entitled Amikacin and Netilmicin Resistance in Pseudomonas aeruginosa.     

Effect of interaction between outer membrane permeability and beta-lactamase production on resistance to beta-lactam agents in gram-negative bacteria

The effect of the interaction of beta-lactamase production and outer membrane permeability on the bacterial resistance to beta-lactam antibiotics was investigated using the Escherichia coli K12 substrains, which have outer membrane mutation and produce TEM-type penicillinase. Experiments confirmed that the cefazolin concentrations in the periplasm of cells exposed to the MIC were restricted to 2.4-4.6 microM among the bacteria, even though the MICs for the bacterial strains were distributed from 13 microM to 1,726 microM. The drug concentrations in the periplasm could be calculated on the basis of parameters obtained experimentally, and the calculated values were in fair agreement with the experimental values. The effect of beta-lactamase activity (Vmax/Km) and the outer membrane barrier on the elevation of MICs was observed to be synergistic, and the contribution of beta-lactamase was more effectively expressed in the bacterial cells with higher outer membrane barrier.     

Resistance of gram-negative bacteria to antibiotics in large calf agglomerations.

The antibiotic resistance of E. coli, Citrobacter, Enterobacter-Klebsiella and Pseudomonas aeruginosa strains isolated from calves was tested. A high proportion of multiresistance was found even in E. coli strains isolated from newborn calves. Gram-negative bacteria isolated from animals in three large calfhouses were almost 100% resistant to ampicillin, tetracyclines and sulphonamides. Multiresistance was general and varied from 5 to 12 antibiotics among different strains. Initial high sensitivity to antibiotics which had never been used before was observed. Antibiotic resistance rapidly increased after use started. The usefulness of antibiotics in E. coli induced diarrhea is questioned and oral rehydration is appraised.     

Long-term antibiotic resistance surveillance of gram-negative pathogens suggests that temporal trends can be used as a resistance warning system

Antibiotic resistance among Gram-negative bacteria and antibiotic consumption were investigated at the Karolinska Hospital, Stockholm, Sweden over a 12-y period. The investigation showed an increase in ciprofloxacin resistance of Escherichia coli from 0% in 1991 to 7% in 1997 and to 11% in 1999. Resistance among Pseudomonas aeruginosa isolates to ciprofloxacin increased from 2.5% in 1991 to 9.0% in 1997 and to 13% in 1999. Resistance levels for norfloxacin showed the same high statistical significance in terms of the temporal trend. A more detailed analysis showed higher resistance against norfloxacin in specific wards. Relationships between antibiotic use and antibiotic susceptibility showed different patterns. The increased ciprofloxacin resistance of E. coli and P. aeruginosa during the study period was paralleled by an increased consumption of quinolones. During the 12-y study period the total use of cephalosporins increased 2.5-fold, while the levels of E. coli resistance to cefuroxime and cefotaxime remained stable. A third pattern was seen with trimethoprim-sulfamethoxazole, namely increasing resistance of E. coli as the use of trimethoprim-sulfamethoxazole declined. The analysis of resistance levels and antibiotic consumption in the present study suggests different mechanisms for the increased resistance. The significant trend of increased resistance to antibiotics over time constitutes an important warning system.     

Comparison of the exoproducts of gram-negative bacteria by SDS-page

The protein exoproducts released during exponential growth of Gram-negative bacteria were analysed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-Page). The following bacterial strains were tested: Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia liquefaciens, Serratia rubidaea, Proteus mirabilis, Proteus vulgaris, Salmonella minnesota, Pseudomonas aeruginosa and Pseudomonas fluorescens. It is demonstrated by SDS-Page that members of one species show identical protein pattern, whereas different species show besides comparable protein bands a species characteristic pattern. All members of Enterobacteriaceae were shown to release proteins whose molecular weights fell into the following size regions: Each strain was shown to synthesize a polypeptide of molecular weight 34,000 and one or more polypeptides within the molecular weight range 25,000–29,000. This profile was shown to be clearly different from that of Pseudomonas strains where 20 or more distinct polypeptides ranging from 12,500 to 160,000 Mr were detectable.     

The elimination of gentamicin-resistant gram-negative bacteria in a newborn intensive care unit

Summary The aminoglycosides play a central role in the treatment of infectious diseases caused by gramnegative bacteria. During the period of January to June 1984, 45 clinical specimens collected in our neonatal intensive care unit grewEnterobacter cloacae; 41 of them were gentamicin resistant. One neonate developed septicemia. The routine antibiotic protocol was then changed from gentamicin-ampicillin to amikacin-ampicillin for a period of six months. During this period the resistance to gentamicin declined to a minimum. Only eight of 122 specimens proved to harbor gram-negative organisms resistant to gentamicin. The gentamicin-resistantE. cloacae vanished. No isolate was resistant to amikacin. The gentamicin-ampicillin regimen was then reintroduced.

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Elimination gentamicinresistenter, gramnegativer Bakterien aus einer Neugeborenen-Intensivstation

Zusammenfassung Aminoglykoside spielen in der Therapie von Infektionen durch gramnegative Bakterien eine zentrale Rolle. Im Zeitraum von Januar bis Juni 1984 wurden aus 45 von Patienten unserer Neugeborenen-Intensivstation entnommenen ProbenEnterobacter cloacae isoliert, davon waren 41 gentamicinresistent. Ein Neugeborenes erkrankte an einer Sepsis. Daraufhin wurde die Routine-Antibiotikatherapie von Gentamicin-Ampicillin für sechs Monate auf Amikacin-Ampicillin umgestellt. In diesem Zeitraum ging die Resistenz gegen Gentamicin auf ein Minimum zurück. Nur aus acht von 122 Proben ließen sich gentamicinresistente gramnegative Mikroorganismen isolieren. Die gentamicinresistentenE. cloacae-Stämme verschwanden. Keines der Isolate war resistent gegen Amikacin. Die Antibiotikatherapie wurde wieder auf Gentamicin-Ampicillin umgestellt.

     

Variation in the propensity to release endotoxin after cefuroxime exposure in different gram-negative bacteria: uniform and dose-dependent reduction by the addition of tobramycin

An aminoglycoside in combination with a beta-lactam antibiotic is often recommended for the treatment of severe infections. The aim of the present study was to study whether cefuroxime-induced endotoxin release could be reduced by addition of tobramycin in different Gram-negative bacteria and how endotoxin release was affected by bacterial killing rate and number of killed bacteria. Three Escherichia coli strains, 1 Klebsiella, 1 Salmonella and 1 Neisseria strain were exposed in vitro to 2, 10 and 50 x minimum inhibitory concentration of cefuroxime, tobramycin or a combination of both. The cefuroxime-induced endotoxin release in the 6 strains varied from 0.1 to 9.9 x 10(-3) EU/killed bacterium. By adding tobramycin, highly significant reductions of 96%, 93%, 97%, 86% and 85% were seen in the 3 E. coli strains and in the Klebsiella and the Salmonella strain, respectively. In the Neisseria strain, the reduction was less. Increasing doses of tobramycin or the combination led to significant endotoxin release reduction in 4/6 strains. In conclusion, addition of tobramycin reduced penicillin-binding protein-3-beta-lactam binding-induced endotoxin release in all tested Gram-negative strains, despite a large interspecies variation in the propensity to release endotoxin. Besides broadening the spectrum and increasing the killing rate, this might be of benefit in the most severe forms of sepsis.