Three Bordetella pertussis typing methods, pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable number tandem repeat analysis (MLVA) were compared using a collection of Swedish strains. Of the three typing methods used, PFGE was found to be the most discriminatory. MLVA and MLST were less discriminatory, but may be valuable for strain discrimination when culture is not possible as they are based on PCR. The combination of MLVA/MLST was found to be equally discriminatory as PFGE and should therefore also be considered. The relationship between predominant lineages in Sweden and The Netherlands, characterized by the PFGE type BpSR11 and the allele for the pertussis toxin promoter ptxP3, respectively, was investigated. Linkage was found between the PFGE type BpSR11 and ptxP3 in that all BpSR11 strains carried ptxP3. On the other hand ptxP3 was found in several other PFGE-types. The presence of the ptxP3 allele in different genetic backgrounds may indicate horizontal gene transfer within B. pertussis or homoplasy. Alternatively, this observation may be due to convergence of PFGE types.
We describe a simple and standardised screening system (AREB) for surveillance of antibiotic resistant bacteria in the environment. The system consists of 96 well microplates containing eight sets of breakpoint amounts of 10 different antibiotics. The incubated microplates are read by a desktop scanner and the plate images are analysed by special software that automatically presents the resistance data. The AREB method is combined with a rapid typing method, the PhenePlate system, which yields information on the diversity of the bacteria in the studied samples, and on the possible prevalence of resistant clones. In order to demonstrate the usage of AREB, a comparative study on the resistance situation among 970 Escherichia coli isolates from sewage and recipient water in Sweden, Norway and Chile, was performed. Resistance rates to all antibiotics were markedly higher in hospital sewage than in other samples. Our data indicate that the AREB system is useful for comparing resistance rates among E. coli and other environmental indicator bacteria in different countries/regions. Simple handling and automatic data evaluation, combined with low cost, facilitate large studies involving several thousands of isolates.
Salmonella enterica, Campylobacter and Yersinia species, Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes and Clostridium perfringens are the bacterial pathogens constituting the greatest burden of food-borne disease in Finland. Several molecular genetic methods have been applied to diagnose, discriminate and survey these bacteria. PCR, PCR-RFLP and PFGE are the most widely and successfully used. However, these methods are unable to replace conventional and internationally standardised phenotyping. Electronic database libraries of the different genomic profiles will enable continuous surveillance of infections and detection of possible infection clusters at an early stage. Furthermore, whole-genome sequence data have opened up new insights into epidemiological surveillance. Laboratory-based surveillance performed in a timely manner and exploiting adequate methods, and co-operation at local, national and international levels are among the key elements in preventing food-borne diseases. This paper reviews different applications of molecular genetic methods for investigating enteric bacterial pathogens and gives examples of the methods successfully used in diagnostics and epidemiological studies in Finland.
The bacterial spectrum in chronic rhinosinusitis (CRS) is clinically relevant. This study aimed to compare two sampling techniques and to characterise Staphylococcus aureus isolated from CRS patients. Bacterial specimens were collected from the nares and maxillary sinus in 42 CRS patients and from the nares in 57 healthy controls. Maxillary sinus sampling was performed in two ways in each patient: with a cotton-tipped aluminium swab through the enlarged sinus ostium, and with a protected brush. S. aureus was characterised by DNA-sequencing of the repeat region of the S. aureus protein A gene, spa typing. The protected brush technique was superior to the cotton-tipped aluminium swab in reducing contamination rate. However, the two sampling methods were consistent in terms of clinically relevant bacterial findings, and the easy-to-handle cotton-tipped swab can still be recommended when culturing the maxillary sinus. Patients showed a significantly higher presence of S. aureus in the nares compared with healthy controls, and healthy controls showed a significantly higher presence of coagulase-negative staphylococci in the nares compared with patients. The spa types were identical for the nares and maxillary sinus in all patients except one. The sampling techniques showed equivalent results, indicating a low risk of unnecessary antibiotic treatment when using the easy-to-handle cotton-tipped aluminium swab. The high rate of identical spa types of S. aureus isolated from the nares and maxillary sinus of CRS patients might indicate colonisation of the maxillary sinus from the nares.
Cites: Rhinology. 2010 Dec;48(4):433-721442080
Cites: Rhinol Suppl. 2012 Mar;(23):3 p preceding table of contents, 1-29822764607
The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Norway is low, but an endemic-like MRSA clone with Staphylococcal protein A (spa)-type t304 has been established especially in nursing homes in the Oslo region causing several large outbreaks. The challenge was that spa-typing and the gold standard Pulsed-Field Gel Electrophoresis (PFGE) were inadequate in discriminating isolates in outbreak investigations. Additional higher resolution genotyping methods were needed. The aims of this study were a) to evaluate whether Multiple-Locus Variable number of tandem repeat Analysis (MLVA) could differentiate within the PFGE clusters between epidemiologically related and unrelated endemic-like ST8-MRSA-IV-t304-PVL-neg (MRSA-t304) isolates and b) investigate the evolution of the endemic-like MRSA-t304 clone over a 15-year time period. All MRSA-t304 isolates detected in the region from 1998 through April 2013 were included. In total, 194 of 197 isolates were available for PFGE and MLVA analyses. PFGE results on isolates from 1998-2010 have been published previously. Two PFGE clusters subdivided into eight MLVA types were detected. One major outbreak clone (PFGE cluster C2/ MLVA type MT5045) appeared from 2004 to 2011 causing long-lasting and large outbreaks in seven nursing homes and one hospital. Five new MLVA types (N = 9 isolates) differing in only one VNTR compared to the outbreak clone C2/MT5045 were detected, but only one (C2/MT5044) was seen after 2011. We suggest that MLVA can replace PFGE analysis, but MLVA may not be the optimal method in this setting as it did not discriminate between all epidemiologically unrelated isolates. The results may indicate that all eight outbreaks in different locations within the PFGE C2 cluster may be branches of one large regional outbreak. The major outbreak strain C2/MT5045 may now, however, be under control, extinguished or has moved geographically.
Cites: J Hosp Infect. 2014 Oct;88(2):72-725085462
Cites: Proc Natl Acad Sci U S A. 2014 May 6;111(18):6738-4324753569
Cites: Infect Control Hosp Epidemiol. 2015 Jul;36(7):777-8525998499
Pasteurella species, widely known as indigenous organisms in the oral and gastrointestinal floras of many wild and domestic animals, are important pathogens in both animals and humans. Human infections due to Pasteurella species are in most cases associated with infected injuries following animal bites. We encountered a rare case of dual infections caused by different two Pasteurella species occurred in a previously healthy 25-year-old female sustaining injury by a dog-bite.
Exudates from the open wound of her dog-bite site, together with the saliva of the dog were submitted for bacteriological examination. Predominantly appearing grayish-white smooth colonies with almost the same colonial properties but slightly different glistening grown on chocolate and sheep blood agar plates were characterized morphologically by Gram's stain, biochemically by automated instrument using Vitek 2 system using GN cards together with commercially available kit system, ID-Test HN-20 rapid panels, and genetically by sequencing the 16S rRNA genes of the organism using a Taq DyeDeoxy Terminator Cycle Sequencing and a model 3100 DNA sequencer instrument.
The causative isolates from the dog-bite site were finally identified as P. canis and P. dagmatis from the findings of the morphological, cultural, and biochemical properties together with the comparative sequences of the 16S rRNA genes. Both the isolates were highly susceptible to many antibiotics and the patient was successfully treated with the administration of so-called the first generation cephalosporin, cefazolin followed by so-called the third generation cephalosporin, cefcapene pivoxil. The isolate from the dog was subsequently identified as P. canis, the same species as the isolate from the patient.
To the best of our knowledge, this was the second report of a dual infection with Pasteurella species consisting of P. dagmatis and P. canis resulting from a dog-bite, followed by the first report of dual infections due to P. dagmatis and P. multocida in 1988. Our isolate finally identified as P. dagmatis was misidentified as P. pneumotripica by means of the Vitek 2 system. The species name "P. dagmatis" was not included in the database of the system. It is also important for routine clinical microbiology laboratories to know the limitation of the automated Vitek 2 system for the accurate identification of Pasteurella species especially P. dagmatis. It should be emphasized that there still exists much room for improvement in Vitek 2 system. Significant improvement of Vitek 2 system especially in the identification of Pasteurella species is urgently desired.
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In 2006 a new variant of Chlamydia trachomatis (nvCT), with a deletion in the cryptic plasmid, was reported in Sweden. This deletion included the targets for the genetic diagnostic systems used in many clinical laboratories and resulted in thousands of false-negative results. The aim of this study was to characterise consecutive Chlamydia tissue culture-positive samples from 2006 in Orebro County, after identification of the nvCT, and to compare the results from samples collected in the same county in 1999-2000. The study also aimed to evaluate the discriminatory capacity of multilocus sequence typing (MLST) compared with ompA sequencing.
ompA sequencing and MLST was used to characterise 100 consecutive Chlamydia tissue culture-positive samples.
In the summer of 1999, the incidence of Salmonella enterica serotype Infantis infections in Alberta rose dramatically. Subsequent laboratory and epidemiological investigations established that an outbreak of human disease caused by this organism was occurring across Canada and was associated with pet treats for dogs produced from processed pig ears. Laboratory investigations using phage typing and pulsed-field gel electrophoresis (PFGE) established that isolates of Salmonella serotype Infantis from pig ear pet treats and humans exposed to pig ear pet treats comprised a well-defined subset of all isolates analyzed. Of the 53 subtypes of Salmonella serotype Infantis obtained around the time of the outbreak as defined by PFGE and phage typing, only 6 subtypes were associated with both human infection and isolation from pig ears. Together with information from epidemiological studies, these investigations established pig ear pet treats as the cause of the Salmonella serotype Infantis outbreak. The results are consistent with a model in which contaminated pig ear pet treats constitute a long-term, continuing vehicle for infection of the human population rather than causing temporally delimited point-source outbreaks. During the course of this outbreak, several other Salmonella serotypes were also isolated from pet treats, suggesting these products may be an important source of enteric infection in both humans and dogs. Though isolates of Salmonella serotypes other than Salmonella serotype Infantis from pet treats were also subjected to PFGE and phage typing, no link with human disease could be definitively established, and the contribution of pig ear pet treats to human disease remains unclear. Elimination of bacterial contamination from pet treats is required to reduce the risk of infection from these products.
Cites: Nord Vet Med. 1984 Sep-Oct;36(9-10):317-236393051
Salmonella enterica spp. enterica serotype Typhimurium (STM) is the most common agent of domestically acquired salmonellosis in Finland. Subtyping methods which allow the characterization of STM are essential for effective laboratory-based STM surveillance and for recognition of outbreaks. This study describes the diversity of Finnish STM isolates using phage typing, antimicrobial susceptible testing, pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis (MLVA), and compares the discriminatory power and the concordance of these methods.
A total of 375 sporadic STM isolates were analysed. The isolates were divided into 31 definite phage (DT) types, dominated by DT1 (47 % of the isolates), U277 (9 % of the isolates) and DT104 (8 % of the isolates). Of all the isolates, 62 % were susceptible to all the 12 antimicrobials tested and 11 % were multidrug resistant. Subtyping resulted in 83 different XbaI-PFGE profiles and 111 MLVA types. The three most common XbaI-PFGE profiles (STYM1, STYM7 and STYM8) and one MLVA profile with three single locus variants accounted for 56 % and 49 % of the STM isolates, respectively. The studied isolates showed a genetic similarity of more than 70 % by XbaI-PFGE. In MLVA, 71 % of the isolates lacked STTR6 and 77 % missed STTR10p loci. Nevertheless, the calculated Simpson's diversity index for XbaI-PFGE was 0.829 (95 % CI 0.792-0.865) and for MLVA 0.867 (95 % CI 0.835-0.898). However, the discriminatory power of the 5-loci MLVA varied among the phage types. The highest concordance of the results was found between XbaI-PFGE and phage typing (adjusted Wallace coefficient was 0.833 and adjusted Rand coefficient was 0.627).
In general, the calculated discriminatory power was higher for genotyping methods (MLVA and XbaI-PFGE) than for phenotyping methods (phage typing). Overall, comparable diversity indices were calculated for PFGE and MLVA (both DI?>?0.8). However, MLVA was phage type dependent providing better discrimination of the most common phage types. Furthermore, 5-loci MLVA was a less laborious method and easier to interpret than XbaI-PFGE. Thus, the laboratory-based surveillance of the Finnish human STM infections has been conducted with a combination of phage typing, antimicrobial susceptibility testing and 5-loci MLVA since January 2014.
Cites: BMC Bioinformatics. 2004 Jan 12;5:414715089
In Finland, occurrence of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has previously been sporadic and related to travel. We describe the first outbreak of colonisation with KPC-KP strain ST512; it affected nine patients in a 137-bed primary care hospital. The index case was detected by chance when a non-prescribed urine culture was taken from an asymptomatic patient with suprapubic urinary catheter in June 2013. Thereafter, all patients on the 38-bed ward were screened until two screening rounds were negative and extensive control measures were performed. Eight additional KPC-KP-carriers were found, and the highest prevalence of carriers on the ward was nine of 38. All other patients hospitalised on the outbreak ward between 1 May and 10 June and 101 former roommates of KPC-KP carriers since January had negative screening results. Two screening rounds on the hospital's other wards were negative. No link to travel abroad was detected. Compared with non-carriers, but without statistical significance, KPC-KP carriers were older (83 vs 76 years) and had more often received antimicrobial treatment within the three months before screening (9/9 vs 90/133). No clinical infections occurred during the six-month follow-up. Early detection, prompt control measures and repetitive screening were crucial in controlling the outbreak.