Factors affecting the measurement of antibiotic resistance in bacteria isolated from lake water

It is more difficult to obtain a reliable assessment of antibiotic resistance in populations of aquatic bacteria than in those populations which are well characterized (e.g. bacteria of medical and veterinary significance). Factors which influence the results include the bacterial taxa involved, their site of origin and the methods and media used to isolate and subculture the bacteria, and to perform the sensitivity tests. Examples of these effects are provided. The resistance profiles obtained with populations of aquatic pseudomonads depend on the species composition of the population. Resistance patterns in aquatic bacteria varied with the site from which they were isolated; a higher incidence of resistance was recorded along shorelines and in sheltered bays than in the open water. The inclusion of antibiotics in the media employed for primary isolation increased the number of individual and multiple resistances recorded. A similar effect was observed with increased inoculum size in the sensitivity disc method but this could be reversed by raising the incubation temperature. The medium used to conduct the test also affected the results and many aquatic bacteria failed to grow on media such as Iso-Sensitest Agar. It is recommended that the sensitivity disc method is adopted for aquatic bacteria because it permits interpretation of a wider range of response. Comparison of the incidence of antibiotic resistance in different habitats will remain meaningless, however, until comprehensive methods for the identification of bacteria are developed and the techniques used for sensitivity testing are standardized.     

Factors affecting the survival and growth of bacteria introduced into lake water

The populations of Pseudomonas sp. B4, Escherichia coli, Klebsiella pneumoniae, Micrococcus flavus, and Rhizobium leguminosarum biovar phaseoli declined rapidly in lake water. The initially rapid decline of the two pseudomonads and R. phaseoli was followed by a period of slow loss of viability, but viable cells of the other species were not found after 10 days. The rapid initial phase of decline was not a result of Bdellovibrio spp., bacteriophages, or toxins in the water since Bdellovibrio spp. were not present and passage of the lake water through filters that should not have removed bacteriophages or soluble toxins led to the elimination of the rapid phase of decline. The addition of 250 g of cycloheximide and 30 g of nystatin per ml eliminated viable protozoa form the lake water, and the population of Pseudomonas sp. B4 did not fall and the decline of E. coli and K. pneumoniae was delayed or slowed under these conditions. Pseudomonas sp. L2 proliferated rapidly in lake water amended with glucose, phosphate, and NH₄NO₃, but its numbers subsequently fell abruptly; however, in water amended with cycloheximide and nystatin, which killed indigenous protozoa, the population density was higher and the fall in numbers was delayed. Of the nutrients, the chief response was to carbon, but when glucose was added, phosphorus and nitrogen stimulated growth further. Removing other bacteria by filtering the lake water before inoculation with Pseudomonas sp. L2 suggested that competition reduced the extent of response of the pseudomonad to added nutrients. We suggest that the decline in lake water of bacteria that are resistant to starvation may be a result of protozoan grazing and that the extent of growth of introduced species may be limited by the supply of available carbon and sometimes of nitrogen and phosphorus, and by predation by indigenous protozoa.     

Monitoring of antibiotic resistance in bacteria isolated from laboratory animals

The drug resistance of microorganisms isolated from laboratory animals never treated with antibiotics is being reported consistently, while the number of laboratory animals used in medicine, pharmacy, veterinary medicine, agriculture, nutrition, and environmental and health science has increased rapidly in Korea. Therefore, this study examined the development of antimicrobial resistance in bacteria isolated from laboratory animals bred in Korea. A total of 443 isolates (7 species) containing 5 Sphingomonas paucimobilis, 206 Escherichia coli, 60 Staphylococcus aureus, 15 Staphylococcus epidermidis, 77 Enterococcus faecalis, 27 Citrobacter freundii, 35 Acinetobacter baumannii were collected from the nose, intestine, bronchus and reproductive organs of ICR mice and SD rats. Of these species, Acinetobacter baumannii and Enterococcus faecalis showed significant antimicrobial resistance according to the minimum inhibition concentration (MIC) in E-test. In case of Acinetobacter baumannii, several isolates showed MIC values 16-128 μg/mL for cefazolin and cefoxitin, and higher resistance (128-512 μg/mL) to nitrofurantoin than that of standard type. Resistance to cefazolin, cefoxitin and nitrofurantoin was detected in 17.14, 20.00, and 8.57% of the Acinetobacter baumannii isolates, respectively. In addition, 44.1% of the Enterococcus faecalis isolates collected from the laboratory animals were resistant to oxacillin concentration of 16-32 μg/mL range, while MIC value of standard type was below oxacillin concentration of 6 μg/mL. These results suggest that in rodent species of laboratory animals, Acinetobacter baumannii are resistance to cefazolin, cefoxitin and nitrofurantoin, whereas those of Enterococcus faecalis were resistance to oxacillin.     

Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking water

Bacterial isolates from the drinking water system of an Oregon coastal community were examined to assess the association of metal tolerance with multiple antibiotic resistance. Positive correlations between tolerance to high levels of Cu2+, Pb2+, and Zn2+ and multiple antibiotic resistance were noted among bacteria from distribution waters but not among bacteria from raw waters. Tolerances to higher levels of Al3+ and Sn2+ were demonstrated more often by raw water isolates which were not typically multiple antibiotic resistant. A similar incidence of tolerance to Cd2+ was demonstrated by isolates of both water types and was not associated with multiple antibiotic resistance. These results suggest that simultaneous selection phenomena occurred in distribution water for bacteria which exhibited unique patterns of tolerance to Cu2+, Pb2+, and Zn2+ and antibiotic resistance.     

Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking water.

Bacterial isolates from the drinking water system of an Oregon coastal community were examined to assess the association of metal tolerance with multiple antibiotic resistance. Positive correlations between tolerance to high levels of Cu2+, Pb2+, and Zn2+ and multiple antibiotic resistance were noted among bacteria from distribution waters but not among bacteria from raw waters. Tolerances to higher levels of Al3+ and Sn2+ were demonstrated more often by raw water isolates which were not typically multiple antibiotic resistant. A similar incidence of tolerance to Cd2+ was demonstrated by isolates of both water types and was not associated with multiple antibiotic resistance. These results suggest that simultaneous selection phenomena occurred in distribution water for bacteria which exhibited unique patterns of tolerance to Cu2+, Pb2+, and Zn2+ and antibiotic resistance.     

Factors influencing the cultivability of lake water bacteria

Counting bacteria in natural water samples by cultivation yields only low recovery efficiencies (ca. 1%), compared to total counts obtained after 4,6-diamidino-2-phenylindol (DAPI) staining. In order to optimize the cultivation of heterotrophic planktonic bacteria from Lake Constance (Germany), selected parameters of the medium composition were modified. The most important factor was the concentration of organic substrate (nutrient broth plus yeast extract), which significantly influenced the "most probable number" obtained in liquid growth medium. Reduced oxygen concentrations (3-12%) lowered the "most probable number". Addition of N-acyl homoserine lactones to the medium increased the cultivability slightly. Low substrate concentrations [0.03-0.06% (w/v)], an incubation atmosphere of 21% oxygen at 16 degrees C for 4 weeks were optimal and increased the cultivability ("most probable number" related to total bacterial counts) to an average cultivability of 18+/-11%, (n=8). The results indicate that cultivabilities of heterotrophic bacteria from lakewater samples can be significantly increased by modifying the cultivation methods.     

Frequency and antibiotic resistance of bacteria isolated from urine cultures]

The AA report the data pertinent to the urine culture performed during a year in the Sanitary and Prophylactic Laboratory of the district of L'Aquila. In urine samples with number of bacteria > 10(5)/ml have been found E. coli (49%), Proteus mirabilis (32%), Pseudomonas aeruginosa (12%), Staphylococcus aureus (5%), Proteus rettgeri (1%), Enterococco (1%). The data are also reported concerning germ resistance to several antibiotics.     

Disinfectant tolerance and antibiotic resistance in psychrotrophic Gram-negative bacteria isolated from vegetables

A total of 330 strains of psychrotrophic non-fermenting Gram-negative bacteria isolated from vegetables were studied. In spite of the wide range of antibiotic resistance occurring, less than 10% showed resistance patterns which included mezclocillin-ticarcillin-gentamicin or ceftizoxime-norfloxacin. Reductions of > 5 log₁₀ in the numbers of cfu were found when these strains were exposed for 30 min to a quaternary ammonium compound (1% w/v).     

Plasmid profile and antibiotic resistance in coagulase-negative staphylococci isolated from polluted water

Four different species of coagulase-negative staphylococci (CNS) were isolated from polluted waters in Fez, Morocco and found to be Staphylococcus simulans, Staph. lenticus, Staph. hyicus and Staph. xylosus . Eight isolates belonging to these four species were analysed for their plasmid content. Southern blot hybridizations were performed to define the resistance determinants of the plasmids harboured by these species. These determinants were found to be carried mainly by Class I staphylococcal plasmids (1–5 kb). A plasmid (4·3 kb) carrying a tetracycline resistance gene was present in five isolates from all identified species. Plasmids carrying a chloramphenicol resistance gene were more frequently encountered and found to be of different sizes. Plasmids carrying erythromycin, neomycin, and streptomycin resistance genes were less frequent and were the same size. The results indicate that the occurrence of multi-resistant CNS in polluted waters may constitute a reservoir for disseminating antibiotic-resistance into the community.     

Factors affecting antagonism of the growth of Phanerochaete chrysosporium by bacteria isolated from soils

Bacteria from polluted and agricultural soils antagonize the growth of Phanerochaete chrysosporium on solid media. The antagonistic bacteria in a soil contaminated with trinitrotoluene included fluorescent pseudomonads. Antagonism by fluorescent pseudomonads was variable according to the pH, and carbon and nitrogen sources used in the growth medium. A fluorescent siderophore produced by a Pseudomonas putida strain did not inhibit the growth of Phanerochaete chrysosporium but pseudomonad isolates capable of producing phenazine derivatives were strongly inhibitory.Utah Agricultural Experiment Station paper no. 4416     

外科血培养标本中病原菌分布及耐药性分析

目的了解中山大学附属第一医院外科血标本中病原菌的菌种分布及常见菌株的耐药性。方法血标本用Bact/A lert-120全自动血培养仪进行血培养,阳性血培养转种后用VITEK-60 AMS细菌鉴定仪鉴定,用K-B法进行药敏试验。结果2002年1月至2005年12月血培养标本中共分离出病原菌256株,阳性率为10.6%。122株(47.7%)为革兰阴性杆菌,其中肠杆菌科细菌占72.1%(88/122),非发酵菌占27.9%(34/122);113株(44.1%)为革兰阳性球菌,其中葡萄球菌属占51.3%(58/113),肠球菌占38.1%(43/113);真菌21株(8.2%)。血培养中的革兰阴性杆菌对亚胺培南、美洛培南治疗敏感;革兰阳性球菌对万古霉素和替考拉宁敏感。结论肠杆菌科细菌和葡萄球菌是外科血培养中的主要病原菌,其耐药现象严重,宜根据药敏结果选用敏感抗菌药物治疗。     

Assessing the anthropogenic impact on lake Shira from antibiotic resistance of heterotrophic bacteria by neural networks methods

A general approach to assessing the anthropogenic impact on lake ecosystems is proposed and exemplified for the case of Lake Shira (Republic of Khakasia, Russia). The impact strength is estimated by applying neural network-based methods to samples of data on interdependent marking features of autochthonous and allochthonous bacteria isolated from the lake in 1997-2001. The proposed combination of analysis methods makes it possible to determine the state of an ecosystem from both small- and large-size samples of data having complex interrelations.     

Natural antibiotic resistance of bacteria isolated from larvae of the oil fly, Helaeomyia petrolei

Helaeomyia petrolei (oil fly) larvae inhabit the asphalt seeps of Rancho La Brea in Los Angeles, Calif. The culturable microbial gut contents of larvae collected from the viscous oil were recently examined, and the majority (9 of 14) of the strains were identified as Providencia spp. Subsequently, 12 of the bacterial strains isolated were tested for their resistance or sensitivity to 23 commonly used antibiotics. All nine strains classified as Providencia rettgeri exhibited dramatic resistance to tetracycline, vancomycin, bacitracin, erythromycin, novobiocin, polymyxin, colistin, and nitrofurantoin. Eight of nine Providencia strains showed resistance to spectinomycin, six of nine showed resistance to chloramphenicol, and five of nine showed resistance to neomycin. All 12 isolates were sensitive to nalidixic acid, streptomycin, norfloxacin, aztreonam, cipericillin, pipericillin, and cefotaxime, and all but OF008 (Morganella morganii) were sensitive to ampicillin and cefoxitin. The oil fly bacteria were not resistant to multiple antibiotics due to an elevated mutation rate. For each bacterium, the number of resistant mutants per 10(8) cells was determined separately on rifampin, nalidixic acid, and spectinomycin. In each case, the average frequencies of resistant colonies were at least 50-fold lower than those established for known mutator strain ECOR 48. In addition, the oil fly bacteria do not appear to excrete antimicrobial agents. When tested, none of the oil fly bacteria produced detectable zones of inhibition on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Candida albicans cultures. Furthermore, the resistance properties of oil fly bacteria extended to organic solvents as well as antibiotics. When pre-exposed to 20 microg of tetracycline per ml, seven of nine oil fly bacteria tolerated overlays of 100% cyclohexane, six of nine tolerated 10% xylene, benzene, or toluene (10:90 in cyclohexane), and three of nine (OF007, OF010, and OF011) tolerated overlays of 50% xylene-50% cyclohexane. The observed correlation between antibiotic resistance and organic solvent tolerance is likely explained by an active efflux pump that is maintained in oil fly bacteria by the constant selective pressure of La Brea's solvent-rich environment. We suggest that the oil fly bacteria and their genes for solvent tolerance may provide a microbial reservoir of antibiotic resistance genes.     

Arthrobacter siderocapsulatus isolated from lake water]

Two microbial strains have been isolated from lake water. The strains oxidize ferrous compounds and manganese. By the structure of microcolonies and the character of deposited oxides of these metals, the strains are identical to the genus Siderocapsa. However, according to their growth cycle and some morpho-physiological characteristics, they were included into the genus Arthrobacter (Corynebacteriaceae). Since these microorganisms differ, by their cultural and morpho-physiological properties, from other species of this genus, they were classed as a new species. Arthrobacter siderocapsulatus nov. sp.     

Combating antibiotic resistance in bacteria

Combinations of certain antibiotics select against resistant strains of bacteria. This finding may provide a strategy of combating antibiotic resistant bacteria.     

Acquired antibiotic resistance in lactic acid bacteria from food

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. -type replicating plasmids of the pAM1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29'871 bp resistance plasmid detected in Lactococcus lacti s subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabo lic traits to generate food-grade vectors.