Repetitive Extragenic Palindromic PCR Research Articles

Analysis of Acquired Resistance Genes in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a gram-negative bacillus and a nosocomial pathogen in immunocompromised patients. Trimethoprim/sulfamethoxazole (TMP/SMX) is the drug of choice for treating S. maltophilia infection; however, resistance to TMP/SMX is increasing. In this study, we investigated the relationship between the incidence of TMP/SMX resistance and the presence of sul genes and mobile elements. A total of 120 S. maltophilia isolates were collected from 3 university hospitals between April 2007 and April 2009. Antimicrobial susceptibilities were determined using the disk diffusion method. PCR and DNA sequencing were conducted for the detection of sul1, sul2, class 1 integron, and ISCR2 element. Repetitive extragenic palindromic sequence-based PCR (REP-PCR) was carried out to evaluate the genetic relatedness. The TMP/SMX-resistant (R) isolates harbored a significantly higher proportion of sul1 gene and class 1 integron than TMP/SMX-susceptible (S) isolates (P<0.001). Seventeen of 28 isolates with sul1 also had a class 1 integron, but none of the isolates without sul1 had a class 1 integron. The identified gene cassettes within class 1 integrons include aacA4, aadA1, aac6'-II, and qac. None of the 120 isolates carried sul2, glmM, or ISCR2 element. REP-PCR did not show any genetic relatedness among the isolates. In Korea, the resistance of S. maltophilia isolates to TMP/SMX is due to sul1 within a class 1 integron rather than to sul2. The class 1 integron also harbors multiple antibiotic resistance genes in addition to sul1, and therefore it could mediate multidrug resistance in S. maltophilia.

Characterization of Strains unlike Mesorhizobium loti That Nodulate Lotus spp. in Saline Soils of Granada, Spain

Lotus species are forage legumes with potential as pastures in low-fertility and environmentally constrained soils, owing to their high persistence and yield under those conditions. The aim of this work was the characterization of phenetic and genetic diversity of salt-tolerant bacteria able to establish efficient symbiosis with Lotus spp. A total of 180 isolates able to nodulate Lotus corniculatus and Lotus tenuis from two locations in Granada, Spain, were characterized. Molecular identification of the isolates was performed by repetitive extragenic palindromic PCR (REP-PCR) and 16S rRNA, atpD, and recA gene sequence analyses, showing the presence of bacteria related to different species of the genus Mesorhizobium: Mesorhizobium tarimense/Mesorhizobium tianshanense, Mesorhizobium chacoense/Mesorhizobium albiziae, and the recently described species, Mesorhizobium alhagi. No Mesorhizobium loti-like bacteria were found, although most isolates carried nodC and nifH symbiotic genes closely related to those of M. loti, considered the type species of bacteria nodulating Lotus, and other Lotus rhizobia. A significant portion of the isolates showed both high salt tolerance and good symbiotic performance with L. corniculatus, and many behaved like salt-dependent bacteria, showing faster growth and better symbiotic performance when media were supplemented with Na or Ca salts.

Bioconservation of Deteriorated Monumental Calcarenite Stone and Identification of Bacteria with Carbonatogenic Activity

The deterioration of the stone built and sculptural heritage has prompted the search and development of novel consolidation/protection treatments that can overcome the limitations of traditional ones. Attention has been drawn to bioconservation, particularly bacterial carbonatogenesis (i.e. bacterially induced calcium carbonate precipitation), as a new environmentally friendly effective conservation strategy, especially suitable for carbonate stones. Here, we study the effects of an in situ bacterial bioconsolidation treatment applied on porous limestone (calcarenite) in the sixteenth century San Jeronimo Monastery in Granada, Spain. The treatment consisted in the application of a nutritional solution (with and without Myxococcus xanthus inoculation) on decayed calcarenite stone blocks. The treatment promoted the development of heterotrophic bacteria able to induce carbonatogenesis. Both the consolidation effect of the treatment and the response of the culturable bacterial community present in the decayed stone were evaluated. A significant surface strengthening (consolidation) of the stone, without altering its surface appearance or inducing any detrimental side effect, was achieved upon application of the nutritional solution. The treatment efficacy was independent of the presence of M. xanthus (which is known as an effective carbonatogenic bacterium). The genetic diversity of 116 bacterial strains isolated from the stone, of which 113 strains showed carbonatogenic activity, was analysed by repetitive extragenic palindromic-polymerase chain reaction (REP-PCR) and 16S rRNA gene sequencing. The strains were distributed into 31 groups on the basis of their REP-PCR patterns, and a representative strain of each group was subjected to 16S rRNA gene sequencing. Analysis of these sequences showed that isolates belong to a wide variety of phylogenetic groups being closely related to species of 15 genera within the Proteobacteria, Firmicutes and the Actinobacteria. This study shows that the abundant carbonatogenic bacteria present in the decayed stone are able to effectively consolidate the degraded stone by producing new calcite (and vaterite) cement if an adequate nutritional solution is used. The implications of these results for the conservation of cultural heritage are discussed.

Molecular characterization of lactic acid bacteria recovered from natural fermentation of beet root and carrot Kanji

The lactic acid bacteria (LAB) play an important role in the fermentation of vegetables to improve nutritive value, palatability, acceptability, microbial quality and shelf life of the fermented produce. The LAB associated with beetroot and carrot fermentation were identified and characterized using different molecular tools. Amplified ribosomal DNA restriction analysis (ARDRA) provided similar DNA profile for the 16 LAB strains isolated from beetroot and carrot fermentation while repetitive extragenic palindromic PCR (rep-PCR) genotyping could differentiate the LAB strains into eight genotypes. Thirteen strains represented by five genotypes could be clustered in five distinct groups while three LAB strains exhibiting distinct genotypes remained ungrouped. These genotypes could be identified to be belonging to L. plantarum group by 16S rDNA sequencing. The recAnested multiplex PCR employing species-specific primers for the L. plantarum group members identified the LAB strains of six genotypes to be L. paraplantarum and the other two genotypes to be L. pentosus. Three genotypes of L. paraplantarum were consistently found on the third and sixth day of beetroot fermentation whereas a distinct genotype of L. paraplantarum and L. pentosus appeared predominant on the tenth day. From carrot Kanji two distinct genotypes of L. paraplantarum and one genotype of L. pentosus were identified. REP-PCR DNA fingerprinting coupled with 16S rDNA sequencing and recA-nested multiplex PCR could clearly identify as well as differentiate the diverse L. plantarum group strains involved in the fermentation.

A comparison of enterococcal genotypes from the human mouth and cheese

Aim The aim of this study was to investigate the prevalence of enterococci in saliva, from within infected root treated teeth, and in British‐produced cheeses. An additional aim was to determine if similar enterococcal genotypes were found within these three groups.Methodology Seventy four samples were collected from the three groups and cultured on Chromocult® enterococci agar. The cultured bacteria were then identified using the Rapid ID 32 Strep kit (bioMerieux). DNA was then extracted from the enterococcal isolates, and genotyping performed by means of repetitive extragenic palindromic (REP) PCR with REP1R‐DT and REP‐2DT primers. Strains were assigned to clonal types by means of the GelCompar software analysis of REP‐PCR profiles.Results Enterococci were isolated from 8 of 50 saliva samples, and from 4 of 15 root canal samples, and from seven of nine cheese samples. Sixteen isolates were identified as E. faecalis, one isolate was identified as E. faecium, one isolate was identified as E. durans, and one isolate identified as E. avium. REP‐PCR revealed nine clonal types within E. faecalis. The same clonal types were found in samples from saliva, from the root canal, and from British‐produced cheeses.Conclusions The prevalence of enterococci in the oral cavity was found to be 12%. The prevalence in the infected previously treated root canal was 27%. Enterococci was isolated from seven out of nine British cheeses investigated. The same clonal types of E. faecalis were recovered from saliva, from in the previously treated infected root canal and from British‐produced cheese's. It would be of interest to investigate the prevalence of E. faecalis in the oral cavity from people that do not eat cheese due to the fact of medical reasons or through choice, to see if there is any difference in the prevalence found.

Isolation, Detection and Genomic Differentiation of Vibrio cholerae and Vibrio parahaemolyticus in Bachok, Kelantan

The objective of this study was to determine the genetic diversity and virulence factors of Vibrio cholerae and V. parahaemolyticus from selected aquatic environments in Bachok, Kelantan, Malaysia. Out of 50 water samples, 33 were confirmed with the presence of presumptive V. cholerae and V. parahaemolyticus based on biochemical tests. Primers targeting toxR gene specific for V. cholerae (800bp) and V. parahaemolyticus (368bp), tdh (251bp) and trh (250bp) virulence genes were used in the PCR. Results of PCR showed that only 25 isolates of V. parahaemolyticus and 3 of V. cholerae had the toxR gene. None of the V. parahaemolyticus isolates had the trh and tdh genes. Repetitive extragenic palindromic PCR (REP-PCR) was used to determine the relatedness of these strains at the genomic level. Cluster analysis for REP-PCR showed 2 major clusters A-B, with several isolates from different locations grouped together indicating the geographical relatedness due to interconnected waterways. REPPCR showed that the species of Vibrios are genetically diverse.

Structure and Diversity of Arsenic Resistant Bacteria in an Old Tin Mine Area of Thailand

The microbial community structure in Thailand soils contaminated with low and high levels of arsenic was determined by denaturing gradient gel electrophoresis (DGGE). Band pattern analysis indicated that the bacterial community was not significantly different in the two soils. Phylogenetic analysis obtained by excising and sequencing six bands indicated that the soils were dominated by Arthobacter koreensis and proteobacteria. Two hundred and sixty-two bacterial isolates were obtained from arsenic contaminated soils. The majority of the As resistant isolates were gram-negative bacteria. MIC studies indicated that all of the tested bacteria had greater resistance to arsenate than arsenite. Some strains were capable of growing in medium containing up to 1,500 mg/l arsenite and arsenate. Correlations analysis of resistance patterns of arsenite resistance indicated that the isolated bacteria could be categorized into 13 groups, with a maximum similarity value of 100%. All strains were also evaluated for resistance to eight antibiotics. The antibiotic resistance patterns divided the strains into 100 unique groups, indicating that the strains were very diverse. Isolates from each antibiotic resistance group were characterized in more detail by using the repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique with ERIC primers. PCR products were analyzed by agarose gel electrophoresis. The genetic relatedness of 100 bacterial fingerprints, determined by using Pearson product moment similarity coefficient, showed that the isolates could be divided into four clusters, with similarity values ranging from 5-99%. While many isolates were genetically diverse, others were clonal in nature Additionally, the arsenic-resistant isolates were examined for the presence of arsenic resistance (ars) genes by using PCR, and 30% of the isolates were found to carry an arsenate reductase encoded by the arsC gene.

Repetitive Extragenic Palindromic PCR (REP-PCR) as an Alternative Method for Detection of Bulking in Activated Sludge

Bulking of activated sludge is a world-wide problem which negatively affects wastewater treatment efficiency. The most common reasons of bulking are bacterial community changes, especially excessive growth of filamentous bacteria (filamentous bulking) or excess of biopolymers on the surface of non-filamentous microbes (non-filamentous or Zoogleal bulking). Because of the complex nature of the bulking phenomenon finding a successful bulking control strategy remains a very important issue that awaits new options and advices. The REP-PCR fingerprinting method has been applied to distinguish a bacterial community in non-bulking and bulking activated sludge. The characteristic REP-PCR fingerprinting patterns were compared with each other in terms of the presence or absence of bands and in terms of measured integrated optical density (IOD) of the bands. The obtained fingerprinting patterns, using Ward's clustering method, have been analyzed to determine homology/similarity relations between specific non-bulking and bulking sludge sampling. The received clustering results were in high concordance with activated sludge typing which generally is done based on physicochemical sludge analysis. The proposed REP-PCR method and statistical analysis of fingerprinting patterns seems to be a simple, rapid and effective method revealing differences between populations in non-bulking and bulking activated sludge. It may be useful for routine activated sludge monitoring and may be helpful in the early detection of the bulking process.

Distribution and diversity of Escherichia coli populations in the South Nation River drainage basin, eastern Ontario, Canada.

We investigated the prevalence and diversity of Escherichia coli strains isolated from surface waters from multiple watersheds within the South Nation River basin in eastern Ontario, Canada. The basin is composed of mixed but primarily agricultural land uses. From March 2004 to November 2007, a total of 2,004 surface water samples were collected from 24 sampling sites. E. coli densities ranged from undetectable to 1.64 x 10(5) CFU 100 ml(-1) and were correlated with stream order and proximity to livestock production systems. The diversity of 21,307 E. coli isolates was characterized using repetitive extragenic palindromic PCR (rep-PCR), allowing for the identification of as many as 7,325 distinct genotypes, without capturing all of the diversity. The community was temporally and spatially dominated by a few dominant genotypes (clusters of more than 500 isolates) and several genotypes of intermediary abundance (clustering between 10 and 499 isolates). Simpson diversity indices, assessed on a normalized number of isolates per sample, ranged from 0.050 to 0.668. Simpson indices could be statistically discriminated on the basis of year and stream order, but land use, discharge, weather, and water physical-chemical properties were not statistically important discriminators. The detection of Campylobacter species was associated with statistically lower Simpson indices (greater diversity; P < 0.05). Waterborne E. coli isolates from genotypes of dominant and intermediary abundance were clustered with isolates obtained from fecal samples collected in the study area over the same period, and 90% of the isolates tested proved to share genotypes with fecal isolates. Overall, our data indicated that the densities and distribution of E. coli in these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne E. coli populations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized E. coli strains.

Repetitive Extragenic Palindromic PCR for Study of Streptococcus mutans Diversity and Transmission in Human Populations

Pulsed-field gel electrophoresis (PFGE) is considered the "gold standard" for molecular epidemiological study. Repetitive extragenic palindromic PCR (rep-PCR) is less time-consuming and more suitable for analyzing large numbers of bacterial strains in human populations. PFGE and rep-PCR provide comparable genotyping results for investigating Streptococcus mutans diversity and transmission.

Phenotypic and Genetic Characterization of Pasteurella Multocida and Related Isolates from Rabbits in Switzerland

Several bacteria belonging to the family Pasteurellaceae are potential pathogens in rabbits. In particular, Pasteurella multocida is considered to be important, and outbreaks caused by this species result in considerable economic losses in rabbitries. However, Pasteurellaceae spp. isolated from rabbits are poorly characterized, and thus, proper identification of P. multocida isolates from these animals is problematic and often unsatisfactory, thereby hampering epidemiological investigations. Therefore, 228 isolates from rabbit populations originating from a breeding and fattening organization with group management and postmortem cases with pasteurellosis from individual owners were phenotypically and genotypically analyzed using biochemical tests and repetitive extragenic palindromic polymerase chain reaction (REP-PCR). Furthermore, 41 samples representing observed phenotypes were selected for phylogenetic analysis using 16S ribosomal RNA and rpoB genes. The REP-PCR typing and phylogenetic analyses correlated well and appeared to be distinct molecular methods for characterization of rabbit isolates. Phenotyping, however, diverged from molecular recognition, reflecting the problematic conventional diagnosis of these strains. The fermentation of sorbitol appeared to be an imprecise indicator for P. multocida subspecies classification. According to REP-PCR and sequencing results, 82% of the isolates were characterized as P. multocida subsp. multocida, 3% as P. multocida subsp. septica, and 5% as P. multocida. Further, 5% were identified as Pasteurella canis. The other 5% represented a homogeneous group of unknown species belonging to the Pasteurellaceae. Samples obtained from individual postmortem cases demonstrated a higher phenotypic and genetic heterogeneity than samples from group management rabbits.

Molecular intraspecific characterization ofPhotobacterium damselaessp.damselaestrains affecting cultured marine fish

The aim of this study was to analyse the intraspecific variability of Photobacterium damselae ssp. damselae strains isolated from different cultured marine fish species using molecular typing methods. Twenty P. damselae ssp. damselae strains isolated from marine fish species were used in this study. Phenotypic characterization of the strains was carried out using standard microbiological methods. Genetic characterization was conducted using three PCR-based methods [random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and repetitive extragenic palindromic-PCR (REP-PCR)]. Dice coefficient and the unweighted pair group method with average linkage were used for numerical analyses of banding patterns. At phenotypic level, the strains analysed showed seven different profiles, which could not be related to the host fish species, geographic area or outbreak of disease. Isolates were grouped into nine and eight clusters using the RAPD technique with primers 5 and 4, respectively. In both cases, the main cluster grouped 45% of strains. The techniques ERIC-PCR and REP-PCR were more discriminatory, both resulting in 14 different clusters, which grouped 15-20% of the isolates. In this study, the techniques tested are confirmed as good tools for molecular typing, because they allow discrimination between P. damselae ssp. damselae strains isolated within the same outbreak. In addition, ERIC-PCR and REP-PCR methods were more adequate for rapid typing of P. damselae ssp. damselae than RAPD, allowing the discrimination at strain level. The results, in agreement with previous studies, confirmed the high intraspecific variability among isolated P. damselae ssp. damselae strains at both phenotypic and genetic levels. This suggests the existence of different clonal lineages that coexist in the same geographic area, within a short period of time (2-3 years). The discrimination at strain level can be useful to study the traceability of infections.

Analysis of the Pan Genome of Campylobacter jejuni Isolates Recovered from Poultry by Pulsed-Field Gel Electrophoresis, Multilocus Sequence Typing (MLST), and Repetitive Sequence Polymerase Chain Reaction (rep-PCR) Reveals Different Discriminatory Capabilities

Campylobacter jejuni is one of the leading bacterial causes of food-borne illness in the USA. Molecular typing methods are often used in food safety for identifying sources of infection and pathways of transmission. Moreover, the identification of genetically related isolates (i.e., clades) may facilitate the development of intervention strategies for control and prevention of food-borne diseases. We analyzed the pan genome (i.e., core and variable genes) of 63 C. jejuni isolates recovered from chickens raised in conventional, organic, and free-range poultry flocks to gain insight into the genetic diversity of C. jejuni isolates recovered from different environments. We assessed the discriminatory power of three genotyping methods [i.e., pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and repetitive extragenic palindromic polymerase chain reaction (rep-PCR)]. The rep-PCR fingerprint was generated by determining the presence of repetitive sequences that are interspersed throughout the genome via repetitive extragenic palindromic PCR, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and BOX element PCR (BOX-PCR) and combining the data to form a composite fingerprint. The genetic fingerprints were subjected to computer-assisted pattern analysis. Comparison of the three genotypic methods revealed that repREB-PCR showed greater discriminatory power than PFGE and MLST. ERIC-PCR and BOX-PCR yielded the highest number of PCR products and greatest reproducibility. Regardless of the genotyping method, C. jejuni isolates recovered from chickens reared in conventional, organic, and free-range environments all exhibit a high level of genotypic diversity.

Specific detection ofLysobacter enzymogenes(Christensen and Cook 1978) strain 3.1T8 with TaqMan®PCR

To develop a strain-specific TaqMan PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. A primer-probe combination was designed on the basis of a strain-specific sequence selected using REP-PCR (repetitive extragenic palindromic-polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer-probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10-fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. The primer-probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. The developed TaqMan PCR method is a strain-specific real-time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy.

Application of a clustering-based peak alignment algorithm to analyze various DNA fingerprinting data

DNA fingerprinting analysis such as amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic PCR (rep-PCR), ribosomal intergenic spacer analysis (RISA), and denaturing gradient gel electrophoresis (DGGE) are frequently used in various fields of microbiology. The major difficulty in DNA fingerprinting data analysis is the alignment of multiple peak sets. We report here an R program for a clustering-based peak alignment algorithm, and its application to analyze various DNA fingerprinting data, such as ARDRA, rep-PCR, RISA, and DGGE data. The results obtained by our clustering algorithm and by BioNumerics software showed high similarity. Since several R packages have been established to statistically analyze various biological data, the distance matrix obtained by our R program can be used for subsequent statistical analyses, some of which were not previously performed but are useful in DNA fingerprinting studies.

CTX-M β-Lactamases inEscherichia colifrom Community-acquired Urinary Tract Infections, Cambodia

Despite the recent global spread of CTX-M beta-lactamases in Escherichia coli isolates from community-acquired urinary tract infections (CA-UTIs), their dissemination has been little studied in developing countries. In a 2-year prospective study, we documented the prevalence of extended-spectrum beta-lactamases (ESBLs) in E. coli that were responsible for CA-UTIs in Phnom-Penh, Cambodia. Ninety-three E. coli strains were included. We observed a high prevalence of resistance to amoxicillin (88.2% of strains), cotrimoxazole (75.3%), ciprofloxacin (67.7%), gentamicin (42.5%), and third-generation cephalosporins (37.7%). A total of 34 strains carried ESBLs, all of which were CTX-M type. CTX-M carriage was associated with resistance to fluoroquinolones and aminoglycosides. U using repetitive extragenic palindromic-PCR, we identified 4 clusters containing 9, 8, 3, and 2 strains. The prevalence of CTX-M beta-lactamases has reached a critical level in Cambodia, which highlights the need for study of their spread in developing countries.

Genetic characterization of Streptococcus phocae strains isolated from Atlantic salmon, Salmo salar L., in Chile

Streptococcus phocae is a beta-haemolytic bacterium frequently involved in disease outbreaks in seals causing pneumonia or respiratory infection. Since 1999, this pathogen has been isolated from diseased Atlantic salmon, Salmo salar, causing serious economic losses in the salmon industry in Chile. In this study, we used different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR) and restriction of 16S-23S rDNA intergenic spacer regions to evaluate the genetic diversity in S. phocae. Thirty-four strains isolated in different years were analysed. The S. phocae type strain ATCC 51973(T) was included for comparative purposes. The results demonstrated genetic homogeneity within the S. phocae strains isolated in Chile over several years, suggesting the existence of clonal relationships among S. phocae isolated from Atlantic salmon. The type strain ATCC 51973(T) presented a different genetic pattern with the PFGE, RAPD, ERIC-PCR and REP-PCR methods. However, the fingerprint patterns of two seal isolates were distinct from those of the type strain.

Nosocomial Outbreak of Infection With Pan–Drug-ResistantAcinetobacter baumanniiin a Tertiary Care University Hospital

To describe what is, to our knowledge, the first nosocomial outbreak of infection with pan-drug-resistant (including colistin-resistant) Acinetobacter baumannii, to determine the risk factors associated with these types of infections, and to determine their clinical impact. Nested case-control cohort study and a clinical-microbiological study. A 1,521-bed tertiary care university hospital in Seville, Spain. Case patients were inpatients who had a pan-drug-resistant A. baumannii isolate recovered from a clinical or surveillance sample obtained at least 48 hours after admission to an intensive care unit (ICU) during the time of the epidemic outbreak. Control patients were patients who were admitted to any of the "boxes" (ie, rooms that partition off a distinct area for a patient's bed and the equipment needed to care for the patient) of an ICU for at least 48 hours during the time of the epidemic outbreak. All the clinical isolates had similar antibiotic susceptibility patterns (ie, they were resistant to all the antibiotics tested, including colistin), and, on the basis of repetitive extragenic palindromic-polymerase chain reaction, it was determined that all of them were of the same clone. The previous use of quinolones and glycopeptides and an ICU stay were associated with the acquisition of infection or colonization with pan-drug-resistant A. baumannii. To control this outbreak, we implemented the following multicomponent intervention program: the performance of environmental decontamination of the ICUs involved, an environmental survey, a revision of cleaning protocols, active surveillance for colonization with pan-drug-resistant A. baumannii, educational programs for the staff, and the display of posters that illustrate contact isolation measures and antimicrobial use recommendations. We were not able to identify the common source for these cases of infection, but the adopted measures have proven to be effective at controlling the outbreak.

Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products

Thirty samples of Italian durum wheat semolina and whole durum wheat semolina, generally used for the production of Southern Italy's traditional breads, were subjected to microbiological analysis in order to explore their lactic acid bacteria (LAB) diversity and to find strains with antifungal activity. A total of 125 presumptive LAB isolates (Gram-positive and catalase-negative) were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and sequence analysis of the 16S rRNA gene, leading to the identification of the following species: Weissella confusa, Weissella cibaria, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus rossiae and Lactobacillus plantarum. The REP-PCR results delineated 17 different patterns whose cluster analysis clearly differentiated W. cibaria from W. confusa isolates. Seventeen strains, each characterized by a different REP-PCR pattern, were screened for their antifungal properties. They were grown in a flour-based medium, comparable to a real food system, and the resulting fermentation products (FPs) were tested against fungal species generally contaminating bakery products, Aspergillus niger, Penicillium roqueforti and Endomyces fibuliger. The results of the study indicated a strong inhibitory activity – comparable to that obtained with the common preservative calcium propionate (0.3% w/v) – of ten LAB strains against the most widespread contaminant of bakery products, P. roqueforti. The screening also highlighted the unexplored antifungal activity of L. citreum, L. rossiae and W. cibaria (1 strain), which inhibited all fungal strains to the same or a higher extent compared with calcium propionate. The fermentation products of these three strains were characterized by low pH values, and a high content of lactic and acetic acids.

Nosocomial Outbreak of Infection With Multidrug-ResistantAcinetobacter baumanniiin a Medical Center in Taiwan

To investigate a nosocomial outbreak of infection with multidrug-resistant (MDR) Acinetobacter baumannii in the intensive care units at China Medical University Hospital in Taiwan. Prospective outbreak investigation. Three intensive care units in a 2,000-bed university hospital in Taichung, Taiwan. Thirty-eight stable patients in 3 intensive care units, all of whom had undergone an invasive procedure, were enrolled in our study. Ninety-four A. baumannii strains were isolated from the patients or the environment in the 3 intensive care units, during the period from January 1 through December 31, 2006. We characterized A. baumannii isolates by use of repetitive extragenic palindromic-polymerase chain reaction (REP-PCR) and random amplified polymorphic DNA (RAPD) fingerprinting. The clinical characteristics of the source patients and the environment were noted. All of the clinical isolates were determined to belong to the same epidemic strain of MDR A. baumannii by the use of antimicrobial susceptibility tests, REP-PCR, and RAPD fingerprinting. All patients involved in the infection outbreak had undergone an invasive procedure. The outbreak strain was also isolated from the environment and the equipment in the intensive care units. Moreover, an environmental survey of one of the intensive care units found that both the patients and the environment harbored the same outbreak strain. The outbreak strain of A. baumannii might have been transmitted among medical staff and administration equipment. Routine and aggressive environmental and equipment disinfection is essential for preventing recurrent outbreaks of nosocomial infection with MDR A. baumannii.