Field Trial of a Microcolony Method for Testing the Antibiotic Sensitivity of Bacteria

The sensitivity of bacteria to antibiotics may be measured by a rapid method in which the criterion of sensitivity is inhibition of microcolony formation on agar which contains antibiotic. As this method allows a report on the antibiotic sensitivity of a pathogenic bacterium four hours after the test is set up, a trial of the method was carried out in a diagnostic laboratory. Two thousand five hundred and four strains of fast-growing bacteria were tested against 10 antibiotics. The overall correlation rate between the four-hour microscopic readings and the subsequent overnight readings on the same cultures was 98.1%. Seven per cent of the microscopical readings attempted gave indeterminate results, and reports on these tests were withheld until the following day.The time spent in making microscopical readings was considered fully justified by the fact that results of a high proportion of antibiotic sensitivity tests were available one day earlier than is usual with established methods.     

A computer system for handling data from bacterial sensitivity testing.

A computer system for handling data collected from antibiotic sensitivity testing of clinical bacterial isolates is described. The system allows a rapid handling of the information collected, supplying the ward and laboratory with cummulative data on each patient. The bacteriologist and the clinician are thus suitably armed when creating a rational antibiotic therapy.     

Evaluation of non-culture diagnosis of invasive meningococcal disease by polymerase chain reaction (PCR)

Antibiotic treatment prior to transport or admission to hospital has reduced the proportion of cases of invasive meningococcal disease (IMD) from which Neisseria meningitidis can be isolated by standard microbiological techniques. Identification of meningococci by polymerase chain reaction (PCR) was assessed in relation to microbiological diagnosis for cases over a 4-year period between 1998 and 2001. A screening assay for the IS1106 gene was used to detect meningococcal DNA and five additional assays for siaD and orf-2 genes were performed to determine the serogroup. PCR results were compared with results of bacteriological culture, other laboratory test results and clinical data. The sensitivity of the PCR assay for culture-confirmed cases was 98.5%. The specificity of the assay was 96% based on test results for patients from whom other bacteria were isolated, children with viral meningitis and afebrile negative controls. The siaD B/C/W-135 and Y as well as the orf-2 gene for serogroup A PCR assays were able to determine the serogroup for 75.2% of cases that were positive by PCR screening assay. When isolates from patients with IMD were tested by both agglutination and PCR, the results agreed in all cases. PCR is a useful tool for diagnosis of IMD when Gram stain and culture tests are negative due to antibiotic treatment prior to collection of samples for microbiological analyses.     

Selection of antibiotic-resistant standard plate count bacteria during water treatment.

Standard plate count (SPC) bacteria were isolated from a drinking-water treatment facility and from the river supplying the facility. All isolates were identified and tested for their resistance to six antibiotics to determine if drug-resistant bacteria were selected for as a consequence of water treatment. Among the isolates surviving our test procedures, there was a significant selection (P less than 0.05) of gram-negative SPC organisms resistant to two or more of the test antibiotics. These bacteria were isolated from the flash mix tank, where chlorine, alum, and lime are added to the water. Streptomycin resistance in particular was more frequent in this population as compared with bacteria in the untreated river water (P less than 0.01). SPC bacteria from the clear well, which is a tank holding the finished drinking water at the treatment facility, were also more frequently antibiotic resistant than were the respective river water populations. When 15.8 and 18.2% of the river water bacteria were multiply antibiotic resistant, 57.1 and 43.5%, respectively, of the SPC bacteria in the clear well were multiply antibiotic resistant. Selection for bacteria exhibiting resistance to streptomycin was achieved by chlorinating river water in the laboratory. We concluded that the selective factors operating in the aquatic environment of a water treatment facility can act to increase the proportion of antibiotic-resistant members of the SPC bacterial population in treated drinking water.     

Effect of age and housing location on antibiotic resistance of fecal coliforms from pigs in a non-antibiotic-exposed herd

The relationship of age and housing location to single antibiotic resistance, multiple antibiotic resistance, and resistance patterns of fecal coliforms obtained during a 20-month period from pigs in a herd that was not exposed to antibiotics for 126 months was determined. Bacteria resistant to single and multiple antibiotics were isolated more frequently (P less than 0.01) from pigs under 7 months of age. A greater proportion of isolates from pigs over 6 months of age was sensitive to the 13 antimicrobial agents tested (P less than 0.01), while a smaller proportion showed resistance to single (P less than 0.05) and multiple (P less than 0.01) antibiotics. More than 80% of the resistant isolates were resistant to tetracycline, streptomycin, or sulfisoxazole. Resistance was greater (P less than 0.01) for pigs in the finishing unit than for those on pasture. Resistance to ampicillin, carbenicillin, and tetracycline was greater (P less than 0.05) for pigs in the finishing unit than for those in the farrowing house. More isolates from pigs on pasture were sensitive to all antimicrobial agents tested (P less than 0.01). A greater proportion of isolates from pigs in the finishing unit showed resistance to a single antibiotic (P less than 0.01). The data from this study suggest that exposure to antibiotics is not the only factor that influences the prevalence of bacteria that are resistant to single and multiple antibiotics in the feces of domestic animals and that considerable research is needed to define the factors influencing antibiotic resistance in fecal bacteria.     

Effect of age and housing location on antibiotic resistance of fecal coliforms from pigs in a non-antibiotic-exposed herd.

The relationship of age and housing location to single antibiotic resistance, multiple antibiotic resistance, and resistance patterns of fecal coliforms obtained during a 20-month period from pigs in a herd that was not exposed to antibiotics for 126 months was determined. Bacteria resistant to single and multiple antibiotics were isolated more frequently (P less than 0.01) from pigs under 7 months of age. A greater proportion of isolates from pigs over 6 months of age was sensitive to the 13 antimicrobial agents tested (P less than 0.01), while a smaller proportion showed resistance to single (P less than 0.05) and multiple (P less than 0.01) antibiotics. More than 80% of the resistant isolates were resistant to tetracycline, streptomycin, or sulfisoxazole. Resistance was greater (P less than 0.01) for pigs in the finishing unit than for those on pasture. Resistance to ampicillin, carbenicillin, and tetracycline was greater (P less than 0.05) for pigs in the finishing unit than for those in the farrowing house. More isolates from pigs on pasture were sensitive to all antimicrobial agents tested (P less than 0.01). A greater proportion of isolates from pigs in the finishing unit showed resistance to a single antibiotic (P less than 0.01). The data from this study suggest that exposure to antibiotics is not the only factor that influences the prevalence of bacteria that are resistant to single and multiple antibiotics in the feces of domestic animals and that considerable research is needed to define the factors influencing antibiotic resistance in fecal bacteria.     

广州地区儿童血培养病原菌的分布及耐药性研究

目的探讨儿童血培养病原菌的分布特点及其耐药情况,为临床诊疗提供参考。方法对广州市儿童医院2005年至2006年临床各科室送检血液标本所分离病原菌的分布及药敏结果进行回顾性分析。结果共检出385株病原菌,其中革兰阳性菌208株,占54．0％,革兰阴性菌164株,占42．6％,真菌13株,占3．4％。分离率前6位的病原菌依次为凝固酶阴性葡萄球菌（CNS,35．8％）、肺炎克雷伯菌（8．8％）、不动杆菌（5．5％）、大肠埃希菌（4．9％）、铜绿假单胞菌（4、7％）、金黄色葡萄球菌（4．4％）。病原菌的病区分布特点：儿科重症监护病房以不动杆菌等非发酵菌为主要分离菌（占41．7％）,新生儿重症监护病房以CNS为主（40．5％）,血液病区以肠杆菌科细菌为主（35．7％）,新生儿病房及传染病房均以CNS为主要分离菌。CNS对青霉素、氨苄西林、红霉素耐药率均超过80％,但对万古霉素、替考拉宁和阿米卡星敏感,MRCNS检出率达72.5％。肠杆菌科细菌对哌拉西林、氨苄西林、头孢噻肟及头孢哌酮的耐药率为50％～100％,但对亚胺培南、阿米卡星和诺氟沙星耐药率较低。不动杆菌对广谱青霉素、第3代头孢菌素、氨曲南及庆大霉素的耐药率较高而对亚胺培南、头孢哌酮／舒巴坦较为敏感。结论凝固酶阴性葡萄球菌是广州地区儿童败血症最主要的病原菌。不同病区检出病原菌种类有较大差异。根据病原菌种类及药敏结果合理应用抗菌药是有效控制感染和减少耐药菌株产生的重要手段。     

Computer-assisted identification of anaerobic bacteria.

A computer program was developed to identify anaerobic bacteria by using simultaneous pattern recognition via a Bayesian probabilistic model. The system is intended for use as a rapid, precise, and reproducible aid in the identification of unknown isolates. The program operates on a data base of 28 genera comprising 238 species of anaerobic bacteria that can be separated by the program. Input to the program consists of biochemical and gas chromatographic test results in binary format. The system is flexible and yields outputs of: (i) most probable species, (ii) significant test results conflicting with established data, and (iii) differential tests of significance for missing test results.     

Computer-assisted identification of anaerobic bacteria.

A computer program was developed to identify anaerobic bacteria by using simultaneous pattern recognition via a Bayesian probabilistic model. The system is intended for use as a rapid, precise, and reproducible aid in the identification of unknown isolates. The program operates on a data base of 28 genera comprising 238 species of anaerobic bacteria that can be separated by the program. Input to the program consists of biochemical and gas chromatographic test results in binary format. The system is flexible and yields outputs of: (i) most probable species, (ii) significant test results conflicting with established data, and (iii) differential tests of significance for missing test results.     

Survey of Shiga toxigenic Escherichia coli O157 and drug-resistant coliform bacteria from in-line milk filters on dairy farms in the Czech Republic

Aims: To determine the occurrence of Shiga toxin‐producing Escherichia coli (STEC) O157 and coliform bacteria isolates resistant to antimicrobial agents in dairy herds by examining milk filters and to analyse the influence of management factors and antibiotic use on antimicrobial resistance. Methods and Results: A total of 192 in‐line milk filters were sampled on 192 dairy farms in the Czech Republic. Information on feeding, husbandry, production, and antibiotic therapy were obtained by questionnaire. The milk filters were cultured for STEC O157 and coliform bacteria. All recovered isolates were examined for antimicrobial susceptibility and presence of antimicrobial‐resistance genes. STEC O157 was detected in four (2%) of the filters. Resistant nonpathogenic E. coli and coliform bacteria isolates with specific genes were detected in 44 (23%) of the filters. Conclusions: The study demonstrated a high prevalence of resistant coliform bacteria in milk filters obtained on Czech dairy farms. Significance and Impact of the Study: The occurrence of resistant coliform bacteria in milk filters was significantly higher among isolates from farms where antibiotic therapy against mastitis was employed during the dry period (P < 0·05).     

Antimicrobial profile of ceftazidime: the situation in three larger hospitals in Prague between 1981 and 1983

As a part of preclinical trials of 2,996 clinical isolates from three larger hospitals in Prague were qualitatively and quantitatively assayed for in vitro sensitivity to ceftazidime. In gramnegative bacteria the incidence of resistance to ceftazidime in strain of Enterobacter, Serratia Proteus and Pseudomonas species ranged from 4% to 6% of strains. In grampositive bacteria only strains of enterococci, listeriae and anaerobic bacteria are excluded from the action of this broad-spectrum antibiotic. According to present experiences the antipseudomonal activity of ceftazidime is approximately the same as that of cefsulodine and cefoperazone. Alarmingly, one of the Pseudomonas aeruginosa isolates was found to show a distinct multiresistance to all available lactam, aminoglycoside and broad-spectrum antimicrobials, including ceftazidime.     

Isolation and characterization of coliforms from glacial ice and water in Canada's High Arctic

Ellesmere Island is the northern most member of the Canadian Arctic Archipelago with over one-third of the land mass covered by ice. A joint services expedition to the island's Blue Mountains offered a unique opportunity for microbiological studies of resident bacteria in an environment uninhabited by man. Over 100 samples of water and ice were collected from stream, lake and glacier and the filtrate cultured under canvas. Bacterial growth was harvested onto swabs for transport back to the UK and 50 coliforms chosen at random for identification and antibiotic susceptibility testing. Most of the glacial strains were capsulated, pigmented and some over 2000 years old. Genera such as Serratia, Enterobacter, Klebsiella and Yersinia were found; speciation was inconclusive and some organisms remain unidentified. Ampicillin resistance was evident in 80% of water isolates as opposed to 30% of the glacial organisms, but the isolates were generally exquisitely susceptible to antibiotics. The facility for ampicillin resistance did not appear to be transferable. Plasmid DNA was found in 33% of the glacial organisms and over 50% of the water isolates. Similar profiles were identified within and apparently between species and required plasmid restriction analysis to help establish identity. Plasmid-free Serratia spp. were subjected to genomic fingerprinting. Indistinguishable patterns were found within sets of isolates both widely spaced by distance and collection date and it was postulated that coliforms able to survive an Arctic environment had spread extensively throughout the expedition area. In conclusion, this study contributes towards knowledge of naturally occurring antibiotic resistance, confirms the presence of plasmids and genotypic data provided evidence that potentially ancient organisms from glaciers can be cultured from water samples significantly distant.     

Copper amendment of agricultural soil selects for bacterial antibiotic resistance in the field

AIMS: The objective of this study was to determine whether Cu-amendment of field plots affects the frequency of Cu resistance, and antibiotic resistance patterns in indigenous soil bacteria. METHODS AND RESULTS: Soil bacteria were isolated from untreated and Cu-amended field plots. Cu-amendment significantly increased the frequency of Cu-resistant isolates. A panel of isolates were characterized by Gram-reaction, amplified ribosomal DNA restriction analysis and resistance profiling against seven antibiotics. More than 95% of the Cu-resistant isolates were Gram-negative. Cu-resistant Gram-negative isolates had significantly higher incidence of resistance to ampicillin, sulphanilamide and multiple (> or =3) antibiotics than Cu-sensitive Gram-negative isolates. Furthermore, Cu-resistant Gram-negative isolates from Cu-contaminated plots had significantly higher incidence of resistance to chloramphenicol and multiple (> or =2) antibiotics than corresponding isolates from control plots. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this field experiment show that introduction of Cu to agricultural soil selects for Cu resistance, but also indirectly selects for antibiotic resistance in the Cu-resistant bacteria. Hence, the widespread accumulation of Cu in agricultural soils worldwide could have a significant effect on the environmental selection of antibiotic resistance.     

人工哺育期腹泻婴猴奇异变形杆菌的分离与鉴定

目的对一株人工哺育期引发恒河猴婴猴腹泻的奇异变形杆菌进行了鉴定,为实验猕猴疾病检测、鉴别诊断提供参考依据。方法通过培养特性、菌落形态、染色、生化试验和血清学诊断鉴别等检查,对分离菌株进行初步鉴定,同时,对分离菌株进行致病性试验及药敏试验。结果通过表型生物学特性鉴定,并结合血清学诊断鉴别方法,确证该分离菌株为奇异变形杆菌,应用药敏试验筛选出了高度敏感的抗菌药,控制了该病的继续发生,致病性试验证明,该分离菌株对小白鼠有高致病性。结论分离到的奇异变形杆菌是导致本次婴猴腹泻死亡的病原菌,该菌为条件致病菌,对实验猕猴和研究人员均有潜在的危害,尽管该菌不是国家标准要求排除的病原菌,但该菌引发的传染病将对动物实验造成严重影响,故应引起高度重视。     

Role of obligate anaerobes in infections in hospitalized patients and therapeutic options

Monitoring of prevalence and antibiotic susceptibility of strictly anaerobic bacteria, causing infections in hospitalized patients, constitutes a part of a program for prudent use of antibiotics. The aim of the study was to assess prevalence of strictly anaerobic bacteria in patients hospitalized in a tertiary care hospital in 2001-2002 with reference to empiric antibiotic therapy. The most common gram-positive bacteria were Clostridium difficile--27.7%, Peptostreptococcus spp. and Peptoniphilus asaccharolyticus--21.9% and Actinomyces spp.--11.1%. There was an increase in the number of stool samples positive for C. difficile toxins A and B from 39.4% in 2001 to 59.0% in 2002. The results of susceptibility testing of gram-positive isolates showed the highest percentages of strains susceptible to piperacilin/tazobactam--99.6%, ticarcillin/clavulanate--98.5%, imipenem--98.5%, amoxicillin/clavulanate--97.4% and piperacillin--97.4%. The most prevalent gram-negative anaerobes were strains of Bacteroides spp.--43.1%, Prevotella spp.--35.8% and Fusobacterium spp.--11.0%. All tested strains of gram-negative bacteria were susceptible to metronidazole, piperacilin/tazobactam, ticarcillin/clavulanate and imipenem. In the analyzed population beta-lactam antibiotics with beta-lactamase inhibitors, carbapenems and metronidazole may be used in empiric therapy of infections caused by strictly anaerobic bacteria.     

Susceptibility testing of anaerobic bacteria: A review of current methods and future prospects

The current NCCLS document, M11 A2, describes two methods for susceptibility testing of anaerobic bacteria. The reference method utilizes an agar dilution procedure, which is labor intensive and not convenient for testing individual patient isolates. The broth microdilution method does not support the growth of 15–40% clinical isolates and demonstrates poor correlation with the reference method for some members of the Bacteroides fragilis group with β-lactam agents and clindamycin. Etest is a new technique that incorporates an antibiotic gradient onto a plastic strip and utilizes agar media. This method is easily performed, permits growth of all anaerobes, and provides quantitative MICs for rapidly growing strains after overnight (20 hr) incubation. This method is convenient and reliable and enables the laboratory to provide the clinician with MIC data for individual patient isolates within a clinically relevant time period.     

The BACTLAB system - a data system for bacteriological routine.

An implemented version of a data system for routine bacteriology is described which uses punch cards to capture all administrative data and OMR (optical mark recognition) documents for the bacteriological findings: diagnosis, antibiotic sensitivity patterns, phage type etc. The output includes reports for the customers and report lists for the laboratory, as well as surveys over findings of pertinent bacteria produced twice each month. In addition bills are produced at regular intervals, both for hospitals and for private patients. All results are stored on magnetic tape in order to make later analysis possible. The system has also been adapted for use in a research project for the study of postoperative infections.     

Bacterial isolates from the bryozoan Membranipora membranacea: influence of culture media on isolation and antimicrobial activity

From specimens of the bryozoan Membranipora membranacea collected in the Baltic Sea, bacteria were isolated on four different media, which significantly increased the diversity of the isolated groups. All isolates were classified according to 16S rRNA gene sequence analysis and tested for antimicrobial properties using a panel of five indicator strains and six different media. Each medium featured a unique set of isolated phylotypes, and a phylogenetically diverse collection of isolates was obtained. A total of 96 isolates were assigned to 49 phylotypes and 29 genera. Only one-third of the members of these genera had been isolated previously from comparable sources. The isolates were affiliated with Alpha- and Gammaproteobacteria, Bacilli, and Actinobacteria. A comparable large portion of up to 22 isolates, i.e., 15 phylotypes, probably represent new species. Likewise, 47 isolates (approximately 50%) displayed antibiotic activities, mostly against grampositive indicator strains. Of the active strains, 63.8 % had antibiotic traits only on one or two of the growth media, whereas only 12.7 % inhibited growth on five or all six media. The application of six different media for antimicrobial testing resulted in twice the number of positive hits as obtained with only a single medium. The use of different media for the isolation of bacteria as well as the variation of media considered suitable for the production of antibiotic substances significantly enhanced both the number of isolates obtained and the proportion of antibiotic active cultures. Thus the approach described herein offers an improved strategy in the search for new antibiotic compounds.     

Identification of virulence factors in Vibrio cholerae isolated from Iraq during the 2007-2009 outbreak

Thousands of people were infected with Vibrio cholerae during the outbreak in Iraq in 2007-2009. Vibrio cholerae was shown to be variable in its content of virulence determinants and in its antibiotic sensitivity. This study was designed to isolate and characterize clinical and environmental V.?cholerae isolates and to determine antibiotic sensitivity, enzyme and toxin production, and the presence of virulence genes. Eighty clinical and five environmental bacterial isolates were collected and diagnosed by subjecting them to microscopic, biochemical, serological, and molecular analysis. The results revealed that 55% of clinical isolates belonged to the Inaba serotype, 32.5% to the Ogawa serotypes, and 12.5% to the Non-O1 serotype. All environmental V.?cholerae isolates belonged to the Non-O1 serotype. All environmental isolates were sensitive to all examined antimicrobial agents, while all clinical isolates showed a high sensitivity (100%) to ampicillin, gentamicin, cephalothin, tetracycline, erythromycin, and ciprofloxacin, and a high resistance (97.5%) to co-trimoxazole, nalidixic acid, and chloramphenicol. It was found that all V.?cholerae (O1) isolates were resistant to the Vibrio static O129 and all Non-O1 V.?cholerae isolates were sensitive to the Vibrio static O129. All clinical and environmental isolates produced hemolysin (100%) and lecithinase (100%), while they showed various production rates of protease (90% of clinical and 60% of environmental) and lipase (50% of clinical and 20% of environmental). The ompW gene was amplified in all the clinical and environmental V.?cholerae isolates, but not in other related and nonrelated bacteria. Multiplex PCR analysis showed that the toxR gene was amplified in all clinical and environmental isolates, while ctxA, ctxB, tcpA genes were amplified only in clinical (O1) isolates. This study indicates the differences in the production of some enzymes and toxins and in the content of virulence genes between clinical and environmental isolates in Iraq during the outbreak (2007-2009).     

Occurrence and patterns of antibiotic resistance in vertebrates off the Northeastern United States coast

The prevalence of antibiotic-resistant bacteria in the marine environment is a growing concern, but the degree to which marine mammals, seabirds and fish harbor these organisms is not well documented. This project sought to identify the occurrence and patterns of antibiotic resistance in bacteria isolated from vertebrates of coastal waters in the northeastern United States. Four hundred and seventy-two isolates of clinical interest were tested for resistance to a suite of 16 antibiotics. Fifty-eight percent were resistant to at least one antibiotic, while 43% were resistant to multiple antibiotics. A multiple antibiotic resistance index value ≥0.2 was observed in 38% of the resistant pathogens, suggesting exposure of the animals to bacteria from significantly contaminated sites. Groups of antibiotics were identified for which bacterial resistance commonly co-occurred. Antibiotic resistance was more widespread in bacteria isolated from seabirds than marine mammals, and was more widespread in stranded or bycaught marine mammals than live marine mammals. Structuring of resistance patterns based on sample type (live/stranded/bycaught) but not animal group (mammal/bird/fish) was observed. These data indicate that antibiotic resistance is widespread in marine vertebrates, and they may be important reservoirs of antibiotic-resistant bacteria in the marine environment.