Simmons Citrate Agar Research Articles

Molecular detection of hlyF gene on multidrug resistance of avian pathogenic Escherichia coli isolated from ducks on wet markets of Surabaya, Indonesia

Abstract. Kendek IA, Putri MFR, Wibisono FJ, Effendi MH, Tyasningsih W, Ugbo EN, Agumah NB. 2024. Molecular detection of hlyF gene on multidrug resistance of avian pathogenic Escherichia coli isolated from ducks on wet markets of Surabaya, Indonesia. Biodiversitas 25: 1246-1252. Avian Pathogenic Escherichia coli (APEC) is a strain of pathogen that can cause colibacillosis in poultry, including ducks worldwide. This disease can be influenced by various virulence genes, one of which is the hlyF gene which can cause systemic disorders and potentially become zoonotic in poultry. This study aimed to detect the hlyF virulence gene from the multidrug resistance (MDR) properties of Escherichia coli originating from 158 duck cloacal swab samples from seven live markets in Surabaya. Samples were isolated and identified using Eosin Methylene Blue Agar (EMBA) and MacConkey Agar (MCA) media, Gram staining, and then continued with biochemical tests of Triple Sugar Iron Agar (TSIA), Simmons Citrate Agar (SCA), Sulfide Indole Motility (SIM), Methyl red (MR) and Voges-Proskauer (VP). Samples were tested for MDR properties and continued with confirmation of the hlyF virulence gene using PCR. Based on the results of this study, it showed that 85% (134/158) were positive for Escherichia coli. Escherichia coli bacteria experienced resistance to the antibiotic erythromycin 96% (129/134), ciprofloxacin 16% (22/134), antibiotics gentamicin 15% (20/134), aztreonam 4.4% (6/134) and chloramphenicol 3% (5/58). The MDR test results in this study were 15% (20/134), while the PCR test results for the hlyF gene were 60% (12/20). Therefore, Escherichia coli was found to have the hlyF virulence gene, an MDR in Escherichia coli bacteria at the Surabaya live market. Furthermore, APEC strains with the hlyF gene, an MDR, potentially affect public health.

Evaluation of culture conditions for sewage-based surveillance of antibiotic resistance in Klebsiella pneumoniae

BackgroundRecent studies have shown promise in predicting clinical antibiotic resistance rates from sewage data. Few have focused on Klebsiella pneumoniae, despite its virulence and importance as carrier of antibiotic resistance. Several media have been suggested for the isolation of K. pneumoniae from complex samples. However, comprehensive evaluations of culture protocols for isolation of K. pneumoniae from sewage are lacking. MethodsHere, influent samples from a major Swedish sewage treatment plant were used to evaluate ten culture conditions in parallel: cultivation on Brilliant green containing Inositol-Nitrate-Deoxycholate agar (BIND), Bruce agar, Klebsiella ChromoSelect Selective agar®, MacConkey-Inositol-Carbenicillin, or Simmons Citrate Agar with Inositol (SCAI) incubated at either 37°C or 42°C for 44 h. The culture conditions were compared based on colony counts of presumed K. pneumoniae and identification precision assessed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. ResultsThe sensitivity was lowest for BIND, whereas it was similar for the other media irrespective of incubation temperature. For four media, a better precision was observed after incubation at 42°C compared to 37°C, to a large extent explained by a lower frequency of captured Klebsiella oxytoca. SCAI incubated at 42°C showed the highest precision (84.4%). By combining this protocol with subsequent antibiotic resistance screening of collected isolates, low resistance rates in sewage K. pneumoniae were revealed, potentially reflecting the local resistance landscape. ConclusionWhen combined with downstream analyses, SCAI incubated at 42°C could be a valuable culture protocol for sewage-based studies on various aspects of K. pneumoniae epidemiology including antibiotic resistance prevalence.

Molecular detection of iron gene on multidrug resistant avian fecal Escherichia coli isolated from broiler on traditional markets, Surabaya, Indonesia

Abstract. Putri MFR, Kendek IA, Wibisono FJ, Effendi MH, Rahardjo D, Tyasningsih W, Ugbo EN. 2023. Molecular detection of iron gene on multidrug resistant avian fecal Escherichia coli isolated from broiler on traditional markets, Surabaya, Indonesia. Biodiversitas 24: 6454-6460. Avian Fecal Escherichia coli (AFEC) is the cause of colibacillosis, which often infects the poultry industry throughout the world. The virulence gene influences the iroN factor, which causes systemic infections in poultry. This research aimed to determine Multidrug Resistance (MDR) in AFEC obtained from traditional markets in Surabaya. There were 96 cloacal swab samples from broiler chickens. The samples were isolated and identified using culture media in the form of Eosin Methylene Blue Agar (EMBA), gram stain, Triple Sugar Iron Agar (TSIA), Sulfide Indole Motility (SIM), Simmons Citrate Agar (SCA), Voges Proskauer (MR-VP) indole and methyl test; detection of multidrug resistance using Mueller-Hinton Agar (MHA) media. The results of the isolation and identification of Escherichia coli bacteria in this study showed that 60.4% of the isolates were positive for Escherichia coli. Escherichia coli is resistant to tetracycline antibiotics by 56%, ciprofloxacin by 55%, antibiotics aztreonam 29%, kanamycin 20%, and chloramphenicol 18%. The multidrug resistance test result on Escherichia coli was 25.8%. The PCR test results for the iroN gene were 40%. Therefore, there are MDR and iroN genes in avian fecal Escherichia coli in Surabaya traditional markets; APEC with iroN gene poses the potential to affect public health.

Population structure and antimicrobial resistance among Klebsiella isolates sampled from human, animal, and environmental sources in Ghana: a cross-sectional genomic One Health study

One Health approaches to address the increasing threat of antimicrobial resistance (AMR) are gaining attention. However, data on the distribution and movement of bacteria and their AMR-associated genes between clinical and non-clinical sources are scarce, especially from low-income and middle-income countries. We aimed to analyse Klebsiella isolates from various sources in Ghana and compare the prevalence of AMR with datasets from two other countries. We conducted a cross-sectional genomic One Health study. Multiple clinical, environmental, and animal sources were sampled from 78 locations (eg, hospitals, residential areas, and farms) in and around Tamale, Ghana. Clinical samples were collected through routine screening and in cases of suspected infection between March 15 and Sept 15, 2019, and samples from the wider environment were collected during a dedicated sampling effort between the dates of Aug 19, 2018, and Sept 26, 2019. Sampling locations were approximately evenly distributed from the centre of the city and steadily outwards to capture both rural and urban locations. Samples with positive growth for Klebsiella were included. Isolates of Klebsiella were obtained from the samples using Simmons citrate agar medium and characterised by antimicrobial susceptibility testing and whole-genome sequencing. A comparative analysis with Klebsiella population surveys from Pavia, Italy, and Tromsø, Norway, was performed. AMR-associated and virulence genes were detected, and the population distribution of these genes was studied. Of 957 samples collected around Tamale, Ghana, 620 were positive for Klebsiella spp. 573 Klebsiella isolates were successfully sequenced, of which 370 were Klebsiella pneumoniae. Only two hospital isolates were carbapenem-resistant. Extended-spectrum β-lactamase (ESBL) genes were relatively common among the Ghanaian clinical isolates but rare in the environmental samples. Prevalence of ESBL genes in human-hospital disease samples was 64% (14 of 22 isolates) in Ghana and 44% (four of nine isolates) in Italy, and prevalence in human-hospital carriage samples was 7% (eight of 107) in Ghana and 13% (54 of 428) in Italy; the prevalence was higher in human-hospital disease samples than in human-hospital carriage samples in both countries, and prevalence across both samples in both countries was higher than in Norway. Ghanaian isolates showed evidence of high recombination rates (recombination events compared with point mutations [r/m] 9·455) and a considerable accessory gene overlap with isolates from Italy and Norway. Although several AMR-associated gene classes were observed relatively frequently in non-clinical sources, ESBL, carbapenemase, and virulence genes were predominantly present only in hospital samples. These results suggest that interventions should be focused on clinical settings to have the greatest effect on the prevalence and dissemination of AMR-associated genes. European Research Council (742158), Academy of Finland EuroHPC grant, Trond Mohn Foundation (BATTALION grant), and Wellcome Trust.

Identification and Pathogenicity of Escherichia coli from Cloacal Swabs

Colibacillosisis is an infectious disease in poultry caused by pathogenic Escherichia coli (E. coli) as primary or secondary agents. This study were aim to identify and test the pathogenicity ofE. coli isolated from chicken cloacal swabs. The samples used in this study were E. coli isolated from chicken cloacal swabs as many as 42 samples. The first stage in this research was subculturing E.coli in Eosin Methylene Blue Agar (EMBA) medium, then identified by Gram stain and biochemistry with Triple Sugar Iron Agar (TSIA), Sulfide Indole Motility (SIM), Simmons Citrate Agar (SCA) and Methyl Red Voges-Proskauer (MRVP). Pathogenicity of E. coli was tested by culturing in Blood Agar to see the results of hemolysin production and on Sorbitol-MacConkey Agar (SMAC) to see the ability of Sorbitol fermentation. The results obtained were 12 isolates was pathogenic E. coli looked from the results of the pathogenicity test in the Blood Agar and SMAC Agar.

Isolation and Detection of Bacterial Strains from Cosmetics Products Available in Pakistan

Cosmetics products are the most essential and frequently used components in our daily life. Besides improving human health, they provide healthy lifestyles and boost our self-esteem. Globally cosmetics market is projected to be 287 billion USD in 2021 to 415 billion USD in 2022. This research study aims at the isolation, identification, and characterization of bacterial strains isolated from cosmetics. Six bacterial colonies were isolated by inoculating different cosmetics products on tryptic soya agar media. All the strains showed optimum growth at 37 °C. All strains were assessed through biochemical tests by using different media such as MacConkey agar, SIM, and Simmons citrate agar and were further proceeded for nucleotide sequencing through Sanger sequencing. Different bacterial strains were revealed in cosmetics products including Sphingomonas paucimobilis, Cytobacillus oceanisediminis, Robertmurraya andreesnii, Cytobacillus firmus, Falsibacillus pallidus, and Acinetobacter junii. Most of these strains were found to be pathogenic however, Sphingomonas has the potential for bioremediation and can be utilized for degrading toxic compounds to make the environment better. Similarly, Cytobacillus is found to be involved in biomineralization and also aids in fermentation. Our results have shown that there is a dire need to assure strict safety regulations regarding cosmetics. Improper manufacturing practices can lead to the contamination of cosmetics which could lead to severe consequences of deteriorating the quality of health. Further studies are needed to explore the potential of these isolates so that they can be utilized to improve our health as well as the environment.

Multidrug resistance-encoding gene in Citrobacter freundii isolated from healthy laying chicken in Blitar District, Indonesia

Background and Aim: The increasing prevalence of resistance (MDR) of Enterobacteriaceae in Indonesia has caused concern regarding human health. Citrobacter freundii reportedly targets the gastrointestinal tract of animals and is a common cause of foodborne diseases associated with diarrhea, peritonitis, meningitis, brain abscess, bacteremia, and urinary tract infection. This study aimed to estimate the prevalence of MDR and the presence of Class 1 integron-encoding genes in C. freundii isolates obtained from cloacal swabs of healthy laying chickens in Blitar district, Indonesia. Materials and Methods: One hundred and sixty-five cloacal swab samples were collected from 33 farms in Blitar over a period of 4 months. Standard microbiological techniques such as bacterial culture in MacConkey agar, Simmons citrate agar, and triple sugar iron agar and biochemical tests such as the indole test were performed to identify the isolates. The antibiotic sensitivity patterns of C. freundii isolates were determined by the disk diffusion method, and MDR-encoding genes (Class 1 integron) were detected by polymerase chain reaction (PCR). Results: Out of 165 cloacal swab samples, 7 (4.24%) were positive for C. freundii. Citrobacter freundii was highly resistant to erythromycin (71.43%) and moderately to streptomycin, tetracycline, and trimethoprim-sulfamethoxazole (all 42.86%); however, it showed low resistance to ampicillin (28.57%). All isolates were found to exhibit MDR. Only 1 (14.29%) of the seven C. freundii isolates harbored a Class 1 integron gene. This study revealed that Class 1 integron-encoding genes have a low prevalence in C. freundii isolated from healthy laying chickens in Blitar, Indonesia. Conclusion: Poultry animals can play a role in the transmission of resistance genes to humans due to the MDR of Enterobacteriaceae, including C. freundii in the intestines.

Biochemical Characterization and Antimicrobial Susceptibility Test of the Bacterial Strain Isolated from Sandwich in Rajshahi University, Bangladesh

This study was conducted to isolate and observe the morphological and biochemical characteristics of bacterial strains present in the sandwich. A single bacterial colony was isolated from a sandwich collected from different restaurants located in the area of the University of Rajshahi by plating from the diluted primary bacterial suspension of the liquid medium onto an agar solidified mineral salt medium after purifying through filter paper. The isolated bacterium was found to be Gram-positive, coccus, motile, lactose-non-fermenting, and could utilize different carbohydrates. Bacterial strain A showed a positive result for the Methyl Red test, the Catalase test, the Indole test, and the Simmons citrate agar test. The optimum culture condition of the isolate was pH 8 and the salt concentration was 0.1 gm/100 ml. The Minimum Inhibitory Concentration (MIC) value against Vancomycin was 50mg/ml and the viable cell count indicated 459×107 CFU/ml. The result showed that the isolated bacterial strain A was resistant to Vancomycin and amoxicillin, whereas it was susceptible to gentamycin, ciprofloxacin, and chloramphenicol. This bacterial strain A can grow to a harmful extent after a certain time of incubation, which may cause a health hazard.

High Prevalence of Klebsiella pneumoniae in European Food Products: a Multicentric Study Comparing Culture and Molecular Detection Methods.

ABSTRACTThe Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices and to characterize food isolates phenotypically and genotypically. Chicken meat (n = 160) and salad (n = 145) samples were collected in five European countries and screened for the presence of KpSC using culture-based and zur-khe intergenic region (ZKIR) quantitative PCR (qPCR) methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44°C for 48 h) was defined as the most suitable selective culture method for KpSC recovery. A high prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and the culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). A total of 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples—same food type and same country (15 cases), different food types and same country (1), and same food type and two countries (1)—suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans.IMPORTANCE Bacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous, and K. pneumoniae is a leading cause of antibiotic-resistant infections in humans. Despite the urgent public health threat represented by K. pneumoniae, there is a lack of knowledge of the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing the presence of KpSC in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies.

Isolation and Identification of Bacteriocin-Producing <i>Bacillus </i>spp from <i>Rastrineobola</i> <i>argentea </i>(Omena) with Activity against Bovine Mastitis Bacterial Pathogens

Rampant use of antibiotics has increased resistance of pathogenic bacteria in both animals and humans. This has triggered the investigation of novel antimicrobial agents produced by a bacterial strain of low virulence with antimicrobial activity with a wide range of clinical significance. This study sought to extract bacteriocins from Bacillus spp that were isolated and identified from Omena (Rastrineobola argentea) and to evaluate the in vitro antimicrobial effect of the obtained bacteriocin against bovine mastitis pathogens. Samples were collected from Lake Victoria using a completely randomized design method followed by isolation of the bacteriocin producing Bacillus spp. Characterization and identification of isolates was done by Gram staining, morphology and Biochemical tests which included catalase, nitrate reduction, methyl Red,-Voges-Proskauer, indole, motility and hydrogen sulphide, growth on Simmons citrate agar, triple sugar iron agar and starch hydrolysis. Antimicrobial activity was done using disc diffusion methods on the nutrient agar and zone of the inhibition measured after 24hours. An analytical profile index system (API 50CH BE, Biomerieux, Inc, France), was also used for identification. Molecular characterization was done by extracting genomic DNA using Qiagen DNA isolation Kit (Qiagen Germany) using the manufacturer's instructions. The gene encoding the 16S rRNA was amplified by PCR using universal bacterial primers pair combination of forwarding primer 27F forward (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R reverse, (5′-GGT TAC CTT GTT ACG ACT T-3′) about Escherichia coli gene sequence. A total of 60 pure isolates were obtained from Omena samples from Lake. Victoria. Morphological characteristics showed varied colour, form, shape and elevation of the pure colonies. About 54 isolates (90%) were gram-positive with spores, while 6 (10%) were gram-negative. Biochemical tests showed varied results among the bacterial isolates. 28 isolates showed antimicrobial activity on E. coli and S. aureus with inhibition measuring between 17-30mm. Analytical profile index system also showed that 20 isolates (71%) were identified as Bacillus subtilis, three isolates (11%) identified as Bacillus pumilus and five isolates (18%) Bacillus mycoides. The Blast analysis of the partial sequences showed100% of the strains of genus Bacillus within the Firmicutes in the domain bacteria. Twenty (20) isolates belong to Bacillus subtilis, Bacillus mycoides represent 18% (5 isolates) while Bacillus pumilus constituted 11% (3 isolates). This study contributes to understanding the use of bacteriocin in the control of diseases in dairy animal farming in Kenya.

Prevalence and Associated Risk Factors of Salmonella, Shigella, and Intestinal Parasites among Food Handlers in Motta Town, North West Ethiopia.

Background Intestinal parasite, Salmonella, and Shigella infections are the main public health concerns in the world, especially in developing countries due to inaccessibility of safe water and unhygienic food handling practices of food handlers. Methods A cross-sectional study was conducted in food handlers in Motta town, from February 2019 to April 2019. The study was undertaken to determine the prevalence and associated risk factors of Salmonella, Shigella, and intestinal parasites among 243 food handlers. Sociodemographic and risk factors were collected using pretested structured questionnaire. The stool sample was collected and examined with direct wet mount and concentration techniques. Culture was performed using xylose lysine deoxycholate agar and MacConkey agar and biochemical tests like Klinger iron agar (KIA), lysine iron agar (LIA), Simmons citrate agar, sulphide indole motility test, citrate utilization, and urease production test were conducted to isolate Salmonella species and Shigella species. An antibiotics susceptibility test was performed with Mueller-Hinton agar using the disk diffusion method. Data were entered using statistical package Epi-Data Version 3.1 and analysed with logistic regression using SPSS version 25 and Fisher's exact test. A p value < 0.05 at 95% CI was considered as statistically significant. Results The prevalence of intestinal parasite, Salmonella, and Shigella was 27.6%, 2.5%, and 1.6%, respectively, and hookworm was the predominant intestinal parasite detected in the stool. Antimicrobial resistance was observed in ampicillin and tetracycline (100%) in Salmonella species and Shigella species. Risk factors like fingernail status, fruit washing before eating, cleaning utensils, and regular shoe wearing habit were associated with intestinal parasite, whereas fingernail status and wearing kitchen gown during food service were significantly associated with Salmonella and Shigella infections. Conclusion The prevalence of intestinal parasitic infections, Salmonella, and Shigella infections in this study indicates the importance of food handlers as probable sources of enteropathogenic infections. Food handlers should have follow-up on the order of food safety rules and keep their personal hygiene. Hotel owners are responsible to control the health status and their created awareness by given food hygiene training for food handlers. Therefore, policy-makers and implementers should focus on the risk factors to reduce the prevalence below the level of public health importance.

Identifikasi Penyakit Bakterial Pada Benih Sidat (Anguilla marmorata) di Balai Budidaya Air Tawar Tatelu

This study aimed to identify the bacterial disease in cultured eel elvers (Anguilla marmorata). The samples used for this study were taken from the Tatelu Freshwater Aquaculture Center. The samples taken were elvers (juvenile life stage of A. marmorata) which showed clinical symptoms of bacterial infection such as bleeding in the chest, abdomen, and the base of the fin, passive movement, weakness, and loss of balance of the body, loss of appetite, some wounds on back fin, chest, and tail, and the body was not slippery/shiny. Three elvers with the size of 4-6 cm, and weigh about 0.2 g were taken as samples. The organ chosen to be targeted for isolation was head kidney. Each sample was isolated twice as replicates and streaked on Triptic Soy Agar (TSA) medium. First replicates were identified in the Tatelu Health Lab of Freshwater Aquaculture, and the second replicates were identified in North Sulawesi Provincial Health Lab. Bacterial identification was done through a series of observations and morphology of bacterial colonies through gram stain, followed by biochemical tests through oxidase test, catalase test, Triple Sugar Iron Agar (TSIA) test, H2S production test, motility test, O/F test, and Simmons Citrate Agar (SCA) test. Water quality parameters that were measured included temperature, disolve oxygen, pH, and ammonia. Based on identification of bacteria in cultured elvers in Tatelu Freshwater Aquaculture Center, the elver samples had been infected with bacterial Aeromonas hydrophila. Keywords: elver, identification, Aeromonas hydrophila, Tatelu Freshwater Aquaculture Center

English

Salmonella is the most important disease causing bacteria in persons as well as in animals. Antimicrobial-resistant Salmonella can be transmitted from animals to humans through consumption of contaminated food and food products. Thus, this study was conducted to determine the prevalence and antimicrobial susceptibility of Salmonella species from lactating cows in dairy farms of Bahir Dar town. A cross-sectional study was conducted in Bahir Dar town from November 2012 to June 2013. Identification of Salmonella spp. was made biochemically with Kliger Iron agar (KIA), Urea agar, Sulfur Indole Motility (SIM), Lysine Deoxycholate agar (LDC) and Simmons citrate agar. Antibacterial sensitivity of the isolates was tested using the Kirby-Bauer test. Thus, out of the total 384 milk samples collected, 36 (9.35%) were positive for Salmonella spp. Salmonella isolates in this study were highly resistant to ampicillin (94.4%) followed by tetracycline (52.8%) and trimethoprim-sulfamethoxazole (38.9%). However, the isolates showed high susceptibility to fluoroquinolones (norfloxacin and ciprofloxacin) at 100 and 93.9%, respectively followed by gentamicin (94.44%). Likewise, the Salmonella isolates showed 72.22% of multidrug-resistance (resistance to two or more antibiotics) in the study area. In conclusion, the incidence of multidrug-resistant Salmonella spp. isolated from milk of lactating cows was high in the study area. Furthermore, the study also revealed the high rate of drug resistance pattern to commonly used antibiotics among the isolated Salmonella spp. Key words: Antimicrobial susceptibility, lactating cows, dairy farm, Salmonella.

The first report of Escherichia fergusonii isolated from non-human primates, in Africa

The aim of this study was to determine the resistance phenotypes of selected enteric bacteria isolated from non-human primates at a wildlife-human interface. Bacterial isolates from faecal samples of non-human primates at two wildlife rehabilitation centres in South Africa were screened for the presence of Escherichia coli. The biochemical characterisation of E. coli and E. coli-like bacteria revealed both adonitol positive and sorbitol negative strains – a unique characteristic of Escherichia fergusonii and Escherichia coli K99. Further tests were carried out to identify the isolates, namely growth on Simmons citrate agar supplemented with 2% adonitol and biochemical tests based on their ability to ferment cellobiose and d-arabitol. Antimicrobial sensitivity was determined with microbroth dilution tests employing microtitre plates with 21 different antimicrobial drugs. Molecular characterisation was done with a duplex polymerase chain reaction (PCR) assay that targeted the yliE and EFER_1569 genes. E. fergusonii strains were confirmed by the presence of a 233bp segment of the yliE gene and a 432bp segment of the EFER_1569 gene.Twenty-three E. coli-like bacteria were confirmed as E. fergusonii based on the confirmatory tests and they were in 100% agreement. Approximately 87% of them were resistant to polymyxins B and E (colistin) as well as the carbapenem group with occasional resistance to amikacin.This is the first reported isolation and identification of E. fergusonii strains in non-human primates. The findings point to E. fergusonii as a possible emerging pathogen of zoonotic importance.

Epidemiology of Klebsiella oxytoca-Associated Diarrhea Detected by Simmons Citrate Agar Supplemented with Inositol, Tryptophan, and Bile Salts

We studied the clinical and epidemiological characteristics of Klebsiella oxytoca-associated diarrhea in hospitalized patients in Hong Kong. Between 1 November 2009 and 30 April 2011, all inositol-fermenting colonies found on Simmons citrate agar supplemented with inositol, tryptophan, and bile salts (SCITB agar) used for the culturing of diarrheal stool samples were screened by a spot indole test for K. oxytoca. The overall sensitivity of SCITB agar plus the spot indole test (93.3%) for the detection of K. oxytoca in stool samples was superior to that of MacConkey agar (63.3%), while the specificities were 100% and 60.4%, respectively. The former achieved a 23-fold reduction in the workload and cost of subsequent standard biochemical identifications. Cytotoxin production and the clonality of K. oxytoca were determined by a cell culture cytotoxicity neutralization assay using HEp-2 cells and pulsed-field gel electrophoresis (PFGE), respectively. Of 5,581 stool samples from 3,537 patients, K. oxytoca was cultured from 117/5,581 (2.1%) stool samples from 104/3,537 (2.9%) patients. Seventy-six of 104 (73.1%) patients with K. oxytoca had no copathogens in their diarrheal stool samples. Twenty-four (31.6%) of 76 patients carried cytotoxin-producing strains, which were significantly associated with antibiotic therapy after hospital admission (50% versus 21.2%; P = 0.01). Health care-associated diarrhea was found in 44 (42%) of 104 patients with K. oxytoca, but there was no epidemiological linkage suggestive of a nosocomial outbreak, and PFGE showed a diverse pattern. None of the patients with cytotoxin-producing K. oxytoca developed antibiotic-associated hemorrhagic colitis, suggesting that K. oxytoca can cause a mild disease manifesting as uncomplicated antibiotic-associated diarrhea with winter seasonality.

IMPROVED MEDIUM FOR DETECTION OF KLEBSIELLA IN POWDERED MILK

ABSTRACT The selectivities to pathogenic Klebsiella strains of different isolation media were compared by known standard strains. The modified MacConkey‐inositol‐carbenicillin (MCIC) medium (Named MCIAC, MacConkey‐inositol‐adonitol‐carbenicillin) supplemented with adonitol gave no false‐negative colonies, and exhibited higher selectivity. MCIC and Simmons citrate agar with inositol (SCAI) media gave two false‐negative colonies, respectively. These three media all gave two false‐positive colonies, respectively. Salmonella Shigella medium gave four false‐negative colonies and five false‐positive colonies. Violet red bile glucose agar medium gave the most false‐positive colonies, although it gave no false‐negative colonies. One hundred samples of powdered milk were examined by MCIAC, MCIC and SCAI plates. The typical positive colonies were further identified using Vitek GNI Auto Microbic system and API 20E system. The results showed that the specificity of the MCIAC medium was higher than MCIC and SCAI media.PRACTICAL APPLICATIONSMacConkey‐inositol‐carbenicillin (MCIC) is the most commonly used selective medium for the detection of Klebsiella. But some inositol‐nonfermenting Klebsiella strains would be missed when selected by this medium. We improved the MCIC medium by supplementing with 1% adonitol. The new modified medium (MacConkey‐inositol‐adonitol‐carbenicillin, MCIAC) had advantages over other selective Klebsiella media in having a higher selectivity and an incubation time of only 16–24 h. MCIAC could be routinely used for pathogenic Klebsiella selection of powdered milk and other food samples.

Primer estudio de Enterobacter sakazakii en alimentos en Cuba

Basándose en ejemplos concretos de sistemas de salud, se aportan elementos y argumentos al debate internacional sobre la importancia de los niveles de atención y de la relación óptima entre niveles. El primer nivel tiene tres funciones esenciales: ser el punto de entrada al sistema, facilitar y coordinar el itinerario de su paciente en el sistema, y asegurar la integración y la síntesis de la información de salud de cada paciente. Describiendo los diferentes obstáculos y problemas posibles, en primer lugar la lógica comercial que se ha ido introduciendo en muchos sistemas, los autores definen puntos de atención y proponen ajustes para asegurar el acceso universal, la equidad, la eficacia y eficiencia, y la sostenibilidad de un sistema de salud.

Genetic and phenotypic analysis of Acinetobacter baumannii insertion derivatives generated with a transposome system.

Acinetobacter baumannii is a metabolically versatile pathogen that causes severe infections in compromised patients. However, little is known about the genes and factors involved in its basic physiology and virulence properties. Insertion mutagenesis was used to initiate the identification and characterization of some of these factors and genes in the prototype strain 19606. The utilization of the pLOFKm suicide delivery vector, which harbors a suicide mini-Tn10 derivative, proved to be unsuccessful for this purpose. The EZ::TN <R6Kgammaori/KAN-2> Tnp transposome system available from Epicentre was then used in conjunction with electroporation to generate isogenic insertional derivatives of A. baumannii 19606. Replica plating showed that 2% of the colonies that grew after electroporation on agar plates without antibiotics also grew in the presence of 40 micro g of kanamycin per ml. DNA hybridization proved that all of the kanamycin-resistant derivatives contained the EZ::TN <R6Kgammaori/KAN-2> insertion element, which was mapped to different genomic locations. Replica plating on Simmons citrate agar and microtiter plate-plastic tube assays identified growth- and biofilm-defective derivatives, respectively. The location of the insertion in several of these derivatives was determined by self-ligation of NdeI- or EcoRI-digested genomic DNA and electroporation of Escherichia coli TransforMax EC100D (pir(+)). Sequence analysis of the recovered plasmids showed that some of the A. baumannii 19606 growth-defective derivatives contain insertions within genes encoding activities required for the generation of energy and cell wall components and for the biosynthesis of amino acids and purines. A gene encoding a protein similar to the GacS sensor kinase was interrupted in four derivatives, while another had an insertion in a gene coding for a hypothetical sensor kinase. A. baumannii 19606 derivatives with defective attachment or biofilm phenotypes had insertions within genes that appear to be part of a chaperone-usher transport system described for other bacteria. DNA hybridization experiments showed that the presence of strain 19606 genes encoding regulatory and attachment or biofilm functions is widespread among other A. baumannii clinical isolates.

New, simple medium for selective recovery of Klebsiella pneumoniae and Klebsiella oxytoca from human feces.

A culture medium was developed which selectively favored the growth of Klebsiella pneumoniae and Klebsiella oxytoca in Escherichia coli-rich fecal cultures, without the use of antibiotics. The discriminative capacity of this medium was based on the presence of only two carbon sources, citrate and inositol, which can be utilized by nearly all K. pneumoniae and K. oxytoca strains but not by E. coli. The medium consisted of Simmons citrate agar (SCA) with 1% inositol (SCAI). Klebsiella strains from fecal samples subcultured on SCAI grew unhampered as yellow, dome-shaped, often mucoid colonies, whereas E. coli appeared as tiny, watery colonies. Apart from some Enterobacter strains, no other types of bacteria were found to mimic the typical appearance of klebsiellae. Recovery experiments from stool samples revealed a limiting ratio of Klebsiella to E. coli of 1:10(6) or more when samples were plated on SCAI versus ratios of 1:10(2) to 1:10(3) on blood agar or Macconkey agar. Compared with an existing Klebsiella culture method, the combination of SCA and MacConkey-inositol-carbenicillin (MIC) agar, Klebsiella yields with SCAI were not lower than those with the combination of MIC and SCA. Furthermore, the efficiency of the SCAI method was twice that of the latter combination. The SCAI plate could be a valuable tool in studies on the epidemiology of K. pneumoniae and K. oxytoca, for example in nosocomial infections, especially those concerning immunocompromised patients.

Milieu sélectif citrate-adonitol pour l'isolement de certaines souches de Escherichia coli K99

Simmons citrate agar supplemented with 0.2% adonitol (SCA medium) was used as a differential medium for some enterotoxigenic Escherichia coli: all strains with either a cryptic K99 antigen or a glucose-dependent K99 antigen and 70% of strains with a glucose-independent K99 antigen gave yellow colonies on SCA medium. Other faecal bacteria did not grow or else developed as blue colonies. Les auteurs proposent un milieu au citrate, selon Simmons, à 0,2% d'adonitol, sur lequel des souches de Escherichia coli à K99 masqué ou glucose-dépendantes et 70% des souches à K99 constitutif forment des colonies jaunes, à la différence de la grande majorité des autres bactéries de la flore entérique saprophyte du veau.