Time-kill study and synergistic activity of cell-wall inhibitor antibiotics in combination with gentamicin against Enterococcus faecalis and Enterococcus faecium

The synergy between gentamicin and vancomycin, teicoplanin, ampicillin and linezolid was studied by time-kill method. Two clinical vancomycin resistant enterococci (VRE) and two vancomycin susceptible enterococci (VSE) isolates were used. Different concentrations of antibiotics were combined. Two VSE strains and the control strain exhibited synergism with the combination of gentamicin, vancomycin, teicoplanin, ampicillin and linezolid. Two VRE strains exhibited synergism with the combination of gentamicin and ampicillin. Synergy between gentamicin and vancomycin, teicoplanin and linezolid was not observed against these isolates. The VRE isolates were positive for vanA, aac (6')-Ie aph (2") and aph (3')-IIIa genes and their vancomycin, teicoplanin and gentamicin MICs were 512 μg/ml, 512 μg/ml and >4000 μg/ml, respectively. In order to treat serious enterococcal infections, further clinical evaluation is needed to examine the in vitro combined effects of gentamicin and vancomycin, teicoplanin and linezolid.     

Different microbial biofilm formation on polymethylmethacrylate (PMMA) bone cement loaded with gentamicin and vancomycin

We studied the in vitro effects of gentamicin and vancomycin alone and in combination added to polymethylmethacrylate (PMMA) cement specimens on the bacterial adhesion of multiresistant clinical isolates.The PMMA specimens (discs) loaded with gentamicin (1.9%) or vancomycin (1.9%) or with a combination of the two were placed in Mueller-Hinton Broth inoculated with bacterial strains. After incubation, bacterial growth was determined by optical density (OD₅₄₀) and sub-cultures. The biofilm PMMA-associated dye (crystal violet) was measured. Antibiotic concentrations in broth were determined by fluorescence polarisation immunoassay.All antibiotic-loaded PMMA cement specimens released high, inhibitory concentrations of gentamicin and vancomycin. However, differences in strain growth and adhesion were recorded. The clinical isolates Met-R/Gent-R CoNS showed no adhesion to gentamicin-loaded specimens for 24 h; strains with Gent-Intermediate susceptibility exhibited growth after 48 h but reduced adhesion. Some Gent-R strains exhibited growth and adhesion to antibiotic-loaded specimens similar to controls (plain discs). Only the VRSA strain (Staphylococcus aureus 5/7) and Escherichia coli were able to grow and adhere to vancomycin-loaded specimens after 24 h of incubation. The specimens loaded with the gentamicin + vancomycin combination showed a synergistic inhibitory effect against all tested strains (no bacterial growth). The degree of bacterial adhesion to PMMA cement loaded with gentamicin or vancomycin may be reduced in spite of a normal growth rate and is different for the tested strains.The effect of gentamicin and vancomycin on bacterial growth and adhesion to PMMA bone cement depends on the antibiotic concentrations, on the characteristics of each specific strain and on its ability to produce biofilm and adhere to antibiotic-loaded PMMA bone cement.     

Bacterial multiresistance to antibiotics. Study of types of bacterial resistance to antibiotics in the environment and in clinical material of a resuscitation ward

Types of resistance were studied in clinical material and in the environment a resuscitation ward in the period from September 1973 to February 1974. Identical strains with the same types of resistance were isolated in both groups. They were verified by qualitative and quantitative tests of sensitivity to selected antibacterial substances. A high degeee of resistance to streptomycin, chloramphenicol, tetracycline and ampicillin was found in all gram-negative bacilli. High effectivity of colimycin, gentamicin and co-trimoxazole was confirmed in vitro. The strains of Staphylococcus aureus were fully sensitive only to oxacillin, lincomycin, spiramycin, vancomycin, gentamicin and co-trimoxazole. The strains of Streptococcus faecalis were found sensitive only to ampicillin and vancomycin.     

Drug resistance of Enterococcus species isolated from the urogenital system

Despite low virulence of enterococci, they have become important nosocomial pathogens. This has been correlated with the increased use of broad-spectrum antibiotics, particularly cephalosporins. Many strains of enterococci exhibit multiple drug resistance; the most important being high-level resistance (HLR) to penicillin (MIC > 100 mg/l) and gentamicin (MIC > 500 mg/l and 2000 mg/l) and/or streptomycin (MIC > 2000 mg/l). The investigation was performed on 92 strains, isolated from genito-urinary tract and recognised as Enterococcus sp. All strains were obtained from several microbiological laboratories of Gdańsk, Gdynia and Tczew. On biochemical reaction profiles species of enterococci were identified as: E. faecalis (72.8%), E. faecalis varians (9.8%), E. durans (7.6%) and E. faecium (9.8%). The minimal inhibitory concentration (MICs) of penicillin, ampicillin, azlocillin, imipenem, gentamicin, amicacin, ciprofioxacin and vancomycin were determined by the agar dilution method. None of these 92 enterococcal strains was vancomycin resistant. 22.2% of E. faecium and 7.5% of E. faecalis showed high-level resistance to penicillin. None of these strains were produced beta-lactamase. High-level resistance to streptomycin and gentamicin was detected. Both--high-level resistance to streptomycin and gentamicin--were found in 6% E. faecalis; 11.1% E. faecalis varians and 22.2% E. faecium.     

Correlation between antibiotic sensitivity testing by conventional and conductivity measurements

A high correlation in the susceptibilities of 44 strains isolated from clinical material to a variety of antimicrobial agents was found between the minimum inhibitory concentration and conductivity measurements. Some discrepancy was found for vancomycin and teicoplanin when tested against 21 strains of staphylococci. Similarly, discrepancies were seen with azlocillin, chloramphenicol and gentamicin when tested against strains of aerobic and facultatively anaerobic Gram-negative bacilli. In the majority of cases in which discrepancies were noted, this reflected differences in relative, and not absolute, susceptibility. With five strains of Pseudomonas however, there was a poor correlation with three of four antibiotics studied. The advantage of this technique is that sensitivities are available within 4–6 h.     

Correlation between antibiotic sensitivity testing by conventional and conductivity measurements

A high correlation in the susceptibilities of 44 strains isolated from clinical material to a variety of antimicrobial agents was found between the minimum inhibitory concentration and conductivity measurements. Some discrepancy was found for vancomycin and teicoplanin when tested against 21 strains of staphylococci. Similarly, discrepancies were seen with azlocillin, chloramphenicol and gentamicin when tested against strains of aerobic and facultatively anaerobic Gram-negative bacilli. In the majority of cases in which discrepancies were noted, this reflected differences in relative, and not absolute, susceptibility. With five strains of Pseudomonas, however, there was a poor correlation with three of four antibiotics studied. The advantage of this technique is that sensitivities are available within 4-6 h.     

In vitro testing of gentamicin-vancomycin loaded bone cement to prevent prosthetic joint infection

Sepsis is a greatly feared complication of total joint arthroplasty. One key question is how to prevent perioperative bacterial adherence, and therefore the potential for infectious complications. The objective of our study was to appraise the emerging capacity of staphylococcal survival on prosthetic materials and to analyze the in vitro effects of gentamicin and vancomycin loaded polymethylmethacrylate (PMMA) cement on bacterial adherence and growth. Hospital acquired staphylococcal strains were systematically inoculated on four orthopedic materials (ultrahigh molecular weight polyethylene, PMMA without antibiotic, commercially produced PMMA loaded with gentamicin, and manually mixed PMMA loaded with gentamicin and vancomycin). Staphylococci were identified using culture and biochemical tests. The inoculated material was allowed to incubate in a liquid broth growth media and subsequently prepared for scanning electron microscopy and bacterial growth quantification. Materials without antibiotics showed evidence of staphylococcal growth. PMMA loaded with only gentamicin grew methicillin-resistant Staphylococcus aureus. Gentamicin-vancomycin loaded PMMA completely inhibited any bacterial growth. Low-dose gentamicin-vancomycin loaded PMMA prevents staphylococcal colonization better than commercially manufactured PMMA loaded with gentamicin. We recommend this combination in high-risk procedures and revision surgeries requiring bone cement.     

Susceptibility to selected chemotherapeutics of Staphylococcus aureus strains resistant to methycyline isolated from clinical materials in the years 1991-1992 and 1997

The susceptibility to selected chemotherapeutic agents was determined in 100 strains of Staphylococcus aureus methicillin-resistant (MRSA) isolated from clinical materials in 1991-1992 (50 strains) and in 1997 (50 strains). Two methods were used for the determination: disc method and antibiotic dilution in agar. The minimal inhibitory concentration (MIC) was determined for vancomycin, teicoplanin, furazolidone, nitrofurantoin, ofloxacin, gentamicin, netilmicin and trimethoprim. The concentrations of the chemotherapeutics in the substrate ranged from 0.125 to 512 mg/l. The obtained results served for drawing of the following conclusions: all studied MRSA strains isolated in 1991-1992 and in 1997 were sensitive to glycopeptide antibiotics: vancomycin and teicoplanin, to nitrofurans: nitrofurantoin and furazolidone, and to fusidic acid. MRSA strains isolated in 1991-1992 were sensitive to ofloxacin, but in 1997 about 80% of the strains were resistant to that antibiotic, and this resistance was noted in S. aureus strains with homogeneous resistance to methicillin. Increasing frequency of resistance to mupirocin was found, in 1991-1992 4% of the strains were resistant, and in 1997 the resistance of MRSA to that antibiotic was found in 12%. No changes occurred in the sensitivity of staphylococci to trimethoprim/sulfamethoxazole (cotrimoxazole). About 94% of strains in 1991-1992 and 1997 were sensitive to that drug. The sensitivity to cotrimoxazole is connected with one of its components (trimethoprim), with 94% of MRSA strains sensitive to it.     

Species-specific sensitivity of coagulase-negative Staphylococci to single antibiotics and their combinations

The activity of beta-lactam antibiotics (oxacillin, cloxacillin, cephalotin), vancomycin, gentamicin and rifampicin applied in vitro individually and in combination against 37 nosocomial methicillin-resistant strains of coagulase-negative staphylococci (CNS) was assessed to demonstrate the heterogeneity of this group of bacteria and estimate the chance of the efficacy of such therapy. The strains belonged to four species: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus cohnii, Staphylococcus hominis. They originated from a hospital environment and from the skin of medical staff of the intensive care unit of a paediatric ward at a university hospital. All strains were methicillin-resistant, according to CLSI standards, but individual strains differed in MIC(ox) values. Susceptibility to other tested antibiotics was also characteristic for the species. The increased susceptibility to antibiotics in combinations, tested by calculating the fractional inhibitory concentration (FIC) index, concerned 26 out of 37 investigated strains and it was a feature of a particular species. Combinations of vancomycin and cephalotin against S. epidermidis and oxacillin with vancomycin were significant, as well as cephalotin and rifampicin in growth inhibition of multiresistant S. haemolyticus strains.     

Antibiotic resistance and virulence factors among clinical and food enterococci isolated in Slovakia

The resistance to antibiotics and the distribution of virulence factors in enterococci isolated from traditional Slovak sheep cheese bryndza was compared with strains from human infections. The occurrence of 4 enterococcal species was observed in 117 bryndza-cheese isolates. The majority of strains were identified as E. faecium (76 %) and E. faecalis (23 %). Several strains of E. durans and 1 strain of E. hirae were also present. More than 90 % of strains isolated from 109 clinical enterococci were E. faecalis, the rest belonged to E. faecium. The resistance to 6 antimicrobial substances (ampicillin, ciprofloxacin, higher concentration of gentamicin, nitrofurantoin, tetracycline and vancomycin) was tested in clinical and food enterococci. A higher level of resistance was found in clinical than in food strains and E. faecium had a higher resistance than E. faecalis; no resistance to vancomycin was detected. The occurrence of 3 virulence-associated genes, cylA (coding for hemolysin), gelE (coding for gelatinase) and esp (coding for surface protein) was monitored. Differences were found in the distribution of cylA gene between clinical and bryndza-cheese E. faecalis strains; in contrast to clinical strains (45 %), cylA gene was detected in 22 % of food isolates. The distribution of 2 other virulence factors, gelE and esp, was not significantly different in the two groups of E. faecalis strains. cylA and gelE genes were not detected in E. faecium but more than 70 % of clinical E. faecium were positive for esp, even thought none of the 79 E. faecium cheese isolates contained this gene.     

Antimicrobial susceptibility, β-lactamase and enterotoxin production in <Emphasis Type="Italic">Bacillus cereus</Emphasis> isolates from clinical and food samples

The antimicrobial susceptibility of 30 clinical and 30 food Bacillus cereus isolates was determined. All isolates were susceptible to streptomycin, ciprofloxacin and gentamicin, 90 % of them to clindamycin and vancomycin, and 67 % to erythromycin. All isolates were resistant to amoxicillin with clavulanic acid, ampicillin, cefotaxime, ciprofloxacin, cloxacillin, cefotaxime with clavulanic acid and penicillin. The MIC values (determined by E-tests) were 48–256 mg/L for ampicillin, 0.19–1.5 mg/L for gentamicin, 0.125–1.0 mg/L for clindamycin, 0.047–4.0 mg/L for erythromycin and 1.5–16 mg/L for vancomycin. The MICs 4.6–18.75 g/L were observed for penicillin using the microdilution method. The presence of metallo-β-lactamases was detected by E-test for 100 % of strains. Nonhemolytic diarrheal enterotoxin (NHE) was produced by 98.3 % of strains, while 31.7 % of them produced hemolytic diarrheal enterotoxin (HBL). Clinical isolates produced 10 % more HBL than food isolates. The psychrotrophic strains isolated from food samples produced NHE at 6.5 °C in 73 % of cases.     

小儿呼吸道感染常见病原菌及药敏结果分析

目的:了解小儿下呼吸道感染的常见病原菌及其对抗菌药物的敏感性.方法:对北京市和平里医院儿科两年来确诊治疗的156例小儿呼吸道感染患儿的痰标本进行培养及药敏试验.结果:156例标本检出致病菌98株,其中G-杆菌71株(72.4％),G+球菌23株(23.5％),真菌4株(4.1％),主要致病菌依次为肺炎克雷伯菌26株,大肠埃希菌20株.流感嗜血杆菌13株,金黄色葡萄球菌7株,肺炎链球菌5株.药敏结果显示,G-杆菌对亚胺培南均敏感,其次为头孢西丁、庆大霉素,G+球菌对万古霉素均敏感,对头孢西丁、庆大霉素、环丙沙星、阿米卡星有较高的敏感性.结论:革兰阴性杆菌为本地区小儿呼吸道感染的主要致病菌,真菌的感染率也呈上升趋势,临床上应及时检测,合理用药.     

Neuroinfections due to Enterococcus faecalis in children

Enterococcal meningitis is a rare complication of neurosurgical procedure or high technology treatment of children and occurs mainly imunocompromised neonates with very low birth weight, severe prematurity and complicates sometime ventriculoperitoneal shunt insertion or perinatal trauma. E. faecalis caused 10 nosocomial meningitis and all strains were susceptible to vancomycin and chloramphenicol, and in our database 90% also to gentamicin and ampicillin. Mortality in our group of 10 children was 20% what is insignificantly higher than overall mortality in the whole cohort of meningitis within last 15 years in our database (15.1%). Early empiric therapy should include also ampicillin or vancomycin, if enterococcal etiology is suspected.     

Survey of antimicrobial resistance in lactic streptococci.

A total of 26 strains of Streptococcus cremoris and 12 strains of Streptococcus lactis were challenged with 18 antimicrobial agents and with nisin in the Bauer-Kirby disk susceptibility test. All strains were susceptible to ampicillin, bacitracin, cephalothin, chloramphenicol, chlortetracycline, erythromycin, penicillin G, tetracycline, and vancomycin. All strains were resistant to trimethoprim, and almost all strains were resistant to sulfathiazole. Variability in resistance to gentamicin, kanamycin, lincomycin, nafcillin, neomycin, nisin, rifampin, and streptomycin was observed. MICs of these substances for the less susceptible strains were determined, and high-level resistance factors could not be detected, except in the case of nisin. S. lactis ATCC 7962 was resistant to at least 40-fold-higher concentrations of nisin (greater than 64 micrograms/ml) than most other strains tested. This strain was a potent nisin producer.     

Survey of antimicrobial resistance in lactic streptococci

A total of 26 strains of Streptococcus cremoris and 12 strains of Streptococcus lactis were challenged with 18 antimicrobial agents and with nisin in the Bauer-Kirby disk susceptibility test. All strains were susceptible to ampicillin, bacitracin, cephalothin, chloramphenicol, chlortetracycline, erythromycin, penicillin G, tetracycline, and vancomycin. All strains were resistant to trimethoprim, and almost all strains were resistant to sulfathiazole. Variability in resistance to gentamicin, kanamycin, lincomycin, nafcillin, neomycin, nisin, rifampin, and streptomycin was observed. MICs of these substances for the less susceptible strains were determined, and high-level resistance factors could not be detected, except in the case of nisin. S. lactis ATCC 7962 was resistant to at least 40-fold-higher concentrations of nisin (greater than 64 micrograms/ml) than most other strains tested. This strain was a potent nisin producer.     

A selective enumeration medium for Carnobacterium maltaromaticum

A selective medium was proposed for isolating the species Carnobacterium maltaromaticum from cheeses. This medium, named CM, was elaborated using combinations of three antibiotics (gentamicin, nalidixic acid, vancomycin) and alkaline pH value (8.8). An experimental design (Doehlert matrix) was drawn up to optimize the experimental conditions of the preparation of the medium. Based on the TS-YE agar medium, it contained 3.5 mg L(-1) of vancomycin, 5.0 mg L(-1) of gentamicin, and 20 mg L(-1) of nalidixic acid. The incubation time was 36 to 48 h at 25 degrees C. The selectivity of this medium was tested against bacterial strains present in the dairy industry and controlled by the PCR method. Thanks to this medium, it was easy to detect C. maltaromaticum and to follow this species in the cheese-making process.     

Antibiotic resistance of Campylobacter and its epidemiologic significance

Sensitivity of 179 strains of thermophilic Campylobacter to 21 chemotherapeutic drugs was studied. Activity of the antibacterial agents against the pathogens was estimated by the MICs. The MIC50 and MIC90 were also determined. All the Campylobacter strains were sensitive to gentamicin, chloramphenicol, neomycin, furazolidone and furagin and resistant to cefazolin, polymyxin E, rifampicin, vancomycin and bacitracin. Differences in the attitude of the Campylobacter isolates from various sources and patients of various age groups to the chemotherapeutic drugs were detected. Possible consideration of the results of comparison of R spectra of Campylobacter strains and the levels of their resistance to antimicrobial drugs as epidemiological markers is discussed.     

Gnom(Cmp): a quantitative approach for comparative analysis of closely related genomes of bacterial pathogens

Comparative genome analysis is a powerful approach to understanding the biology of infectious bacterial pathogens. In this study, a quantitative approach, referred to as Gnom(Cmp), was developed to study the microevolution of bacterial pathogens. Although much more time-consuming than existing tools, this procedure provides a much higher resolution. Gnom(Cmp) accomplishes this by establishing genome-wide heterogeneity genotypes, which are then quantified and comparatively analyzed. The heterogeneity genotypes are defined as chromosomal base positions that have multiple variants within particular genomes, resulted from DNA duplications and subsequent mutations. To prove the concept, the procedure was applied on the genomes of 15 Staphylococcus aureus strains, focusing extensively on two pairs of hVISA/VISA strains. hVISA refers to heteroresistant vancomycin-intermediate S. aureus strains and VISA is their VISA mutants. hVISA/VISA displays some remarkable properties. hVISA is susceptible to vancomycin, but VISA mutants emerge soon after a short period of vancomycin therapy, therefore making the pathogen a great model organism for fast-evolving bacterial pathogens. The analysis indicated that Gnom(Cmp) could reveal variants within the genomes, which can be analyzed within the global genome context. Gnom(Cmp) discovered evolutionary hotspots and their dynamics among many closely related, even isogenic genomes. The analysis thus allows the exploration of the molecular mechanisms behind hVISA/VISA evolution, providing a working hypotheses for experimental testing and validation.     

耐甲氧西林凝固酶阴性葡萄球菌中多种抗生素耐药基因的观察

探讨耐甲氧西林凝固酶阴性葡萄球菌(methicillin-resistant coagulase negative staphylococci,MRCNS)中多种抗生素耐药基因的分布情况,采取Kirby Bauer标准纸片扩散法(K-B纸片扩散法)对MRCNS开展药敏实验,并通过PCR检测菌种携带的抗生素耐药基因。药敏实验结果显示,96株MRCNS对庆大霉素、红霉素、莫匹罗星以及四环素的耐药性各有差异,但对氨苄西林的耐药性却达到100%,未发现对万古霉素具有耐药性的菌株;PCR检测结果显示,9种耐药基因merA、ermB、ermC、msrA、tetM、tetK、aac(6’)-aph(2")、aph(3’)-III、mupA的阳性检出率各有差异,没有扩增出vanA、ermA、vanB以及ant(6’)-I基因,但都扩增出mecA基因。结果表明,MRCNS可以同时携带多种抗生素耐药基因,属于一种多重耐药菌。     

Combined antimicrobial resistance in Enterococcus faecium isolated from chickens

Nineteen E. faecium strains isolated from chicken caecum samples, collected in slaughterhouses and highly resistant to vancomycin or gentamicin, were coresistant to erythromycin, and/or tetracyclines, and/or streptogramins, and/or avilamycin. Multiple antibiotic resistance was related to the presence in various combinations of aac(6')-aph(2"), erm(B), emtA, mef(A), tet(L), tet(M), and vanA genes.