Amplified Fragment Length Polymorphism Typing Research Articles

The first established microsatellite markers to distinguish Candida orthopsilosis isolates and detection of a nosocomial outbreak in China.

The infection proportion of Candida orthopsilosis, a member of the C. parapsilosis complex, has increased globally in recent years, and nosocomial outbreaks have been reported in several countries. This study aimed to establish microsatellite loci-based typing method that was able to effectively distinguish among C. orthopsilosis isolates. Three reference C. orthopsilosis genome sequences were analyzed to identify repeat loci. DNA sequences containing over eight bi- or more nucleotide repeats were selected. A total of 51 loci were initially identified, and locus-specific primers were designed and tested with 20 epidemiologically unrelated isolates. Four loci with excellent reproducibility, specificity, and resolution for molecular typing purposes were identified, and the combined discriminatory power (DP, based on 20 epidemiologically unrelated isolates) of these four loci was 1.0. Reproducibility was demonstrated by consistently testing three strains each in triplicate, and stability, demonstrated by testing 10 successive passages. Then, we collected 48 C. orthopsilosis non-duplicate clinical isolates from the China Hospital Invasive Fungal Surveillance Net study to compare the DP of the microsatellite-based typing with internal transcribed spacer (ITS) and amplified fragment length polymorphism (AFLP) typing analyses, using ATCC 96139 as a reference strain. These 49 isolates were subdivided into 12 microsatellite types (COMT1-12), six AFLP types, and three ITS types, while all the isolates with the same COMT belonged to consistent AFLP and ITS type, demonstrating the high DP of our microsatellite-type method. According to our results, COMT12 was found to be the predominant type in China, and COMT5 was the second largest and responsible for causing a nosocomial outbreak. This microsatellite-type method is a valuable tool for the differentiation of C. orthopsilosis and could be vital for epidemiological studies to determine strain relatedness and monitor transmission.

First Report of Candidemia Clonal Outbreak Caused by Emerging Fluconazole-Resistant Candida parapsilosis Isolates Harboring Y132F and/or Y132F+K143R in Turkey.

Clonal outbreaks of fluconazole-resistant (FLZR) Candida parapsilosis isolates have been reported in several countries. Despite its being the second leading cause of candidemia, the azole resistance mechanisms and the clonal expansion of FLZR C. parapsilosis blood isolates have not been reported in Turkey. In this study, we consecutively collected C. parapsilosis blood isolates (n = 225) from the fifth largest hospital in Turkey (2007 to 2019), assessed their azole susceptibility pattern using CLSI M27-A3/S4, and sequenced ERG11 for all and MRR1, TAC1, and UPC2 for a selected number of C. parapsilosis isolates. The typing resolution of two widely used techniques, amplified fragment length polymorphism typing (AFLP) and microsatellite typing (MST), and the biofilm production of FLZR isolates with and without Y132F were compared. Approximately 27% of isolates were FLZR (60/225), among which 90% (54/60) harbored known mutations in Erg11, including Y132F (24/60) and Y132F+K143R (19/60). Several mutations specific to FLZR isolates were found in MRR1, TAC1, and UPC2 AFLP grouped isolates into two clusters, while MST revealed several clusters. The majority of Y132F/Y132F+K143R isolates grouped in clonal clusters, which significantly expanded throughout 2007 to 2019 in neonatal wards. Candida parapsilosis isolates carrying Y132F were associated with significantly higher mortality and less biofilm production than other FLZR isolates. Collectively, we documented the first outbreak of FLZR C. parapsilosis blood isolates in Turkey. The MRR1, TAC1, and UPC2 mutations exclusively found in FLZR isolates establishes a basis for future studies, which will potentially broaden our knowledge of FLZR mechanisms in C. parapsilosis MST should be a preferred method for clonal analysis of C. parapsilosis isolates in outbreak scenarios.

PME and Other ESBL-Positive Multiresistant Pseudomonas aeruginosa Isolated from Hospitalized Patients in the Region of Kurdistan, Iraq.

Nosocomial infections occur worldwide and also in the Kurdistan region. Frequently patients colonized with multiresistant Pseudomonas aeruginosa isolates are encountered in many hospitals. As information is lacking with respect to the mechanisms of resistance responsible for the multiresistant character of the P. aeruginosa isolates and their genetic relationship, isolates were prospectively collected and characterized with respect to their mechanism of resistance. During 2012 and 2013, 81 P. aeruginosa isolates were collected from three teaching hospitals in the city of Erbil, Iraq. Susceptibility testing was performed using the VITEK-2 system. Isolates were screened for the presence of extended-spectrum β-lactamases (ESBLs) and for the presence of metallo β-lactamases (MBLs). The presence of serine carbapenemases was detected by PCR. The genetic relationship of the isolates was demonstrated by amplified fragment length polymorphism (AFLP). Susceptibility results revealed high rates of resistance against all classes of antibiotics except polymyxins. Genetic characterization demonstrated the presence of ESBL-genes, that is, blaVEB (30%) and blaPER (17%), also ESBL blaPME was detected in four isolates. AFLP typing revealed clonal spread of blaVEB, blaPER, and three clusters of blaOXA-10-positive isolates. Only one isolate was MBL (blaVIM) positive. Of a selected number of isolates (n = 11), whole-genome sequencing analysis revealed that these isolates belonged to "high-risk" MLSTs ST244, ST235, ST308, and ST654. This study reveals the presence and clonal spread of widely resistant high-risk clones of P. aeruginosa in Iraqi Kurdistan. As far as we are aware, this is the first report of multiple, polyclonal, PME producing P. aeruginosa outside the Arabian Peninsula.

Whole genome sequencing of emerging multidrug resistant Candida auris isolates in India demonstrates low genetic variation.

Candida auris is an emerging multidrug resistant yeast that causes nosocomial fungaemia and deep-seated infections. Notably, the emergence of this yeast is alarming as it exhibits resistance to azoles, amphotericin B and caspofungin, which may lead to clinical failure in patients. The multigene phylogeny and amplified fragment length polymorphism typing methods report the C. auris population as clonal. Here, using whole genome sequencing analysis, we decipher for the first time that C. auris strains from four Indian hospitals were highly related, suggesting clonal transmission. Further, all C. auris isolates originated from cases of fungaemia and were resistant to fluconazole (MIC >64 mg/L).

Comparison of MALDI-TOF MS and AFLP for strain typing of ESBL-producing Escherichia coli

Typing of bacterial isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) potentially provides an efficient on-site method to monitor the spread of antibiotic-resistant bacteria and rapidly detect outbreaks. We compared MALDI-MS typing results to those of amplified fragment length polymorphism (AFLP) in a collection of 52 ESBL-producing Escherichia coli, isolated in a Dutch nursing home with an on-going outbreak of ST131 E. coli. Specific MALDI types were defined based on spectral data from four replicate colony samples of isolates grown on Columbia agar using multivariate statistical procedures. Type-specific superspectra were computed for four E .coli MALDI-types and tested for the potential of rapid and automated typing. The effect of different incubation conditions on typing performance was tested by analysing five isolates incubated for 24 h and 48 h on five different media. Types defined based on MALDI spectra were largely in agreement with the AFLP results, although some MALDI types comprised of more than one AFLP type. In particular, isolates belonging to ST131 showed distinct mass patterns. The proportion of isolates correctly assigned was substantially lower for isolates incubated on Sabouraud-dextrose and Drigalski agars for 24 h, and for those incubated for 48 h (all media). Our results show that the identification of type-specific peaks potentially allows direct typing of isolates belonging to specific clonal lineages. Both incubation time and media affected type assignment, suggesting that there is a need for a careful standardization of incubation time and culturing conditions when developing MALDI-typing schemes for E. coli.

Evidence of genotypic diversity among Candida auris isolates by multilocus sequence typing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and amplified fragment length polymorphism

Candida auris is a multidrug-resistant nosocomial bloodstream pathogen that has been reported from Asian countries and South Africa. Herein, we studied the population structure and genetic relatedness among 104 global C. auris isolates from India, South Africa and Brazil using multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). RPB1, RPB2 and internal transcribed spacer (ITS) and D1/D2 regions of the ribosomal DNA were sequenced for MLST. Further, genetic variation and proteomic assessment was carried out using AFLP and MALDI-TOF MS, respectively. Both MLST and AFLP typing clearly demarcated two major clusters comprising Indian and Brazilian isolates. However, the South African isolates were randomly distributed, suggesting different genotypes. MALDI-TOF MS spectral profiling also revealed evidence of geographical clustering but did not correlate fully with the genotyping methods. Notably, overall the population structure of C. auris showed evidence of geographical clustering by all the three techniques analysed. Antifungal susceptibility testing by the CLSI microbroth dilution method revealed that fluconazole had limited activity against 87% of isolates (MIC90, 64 mg/L). Also, MIC90 of AMB was 4 mg/L. Candida auris is emerging as an important yeast pathogen globally and requires reproducible laboratory methods for identification and typing. Evaluation of MALDI-TOF MS as a typing method for this yeast is warranted.

Phylogeography and evolutionary patterns in Sporothrix spanning more than 14 000 human and animal case reports.

Pathology to vertebrate hosts has emerged repeatedly in the order Ophiostomatales. Occasional infections have been observed in Sporothrix mexicana at a low level of virulence, while the main pathogenic species cluster in a derived clade around S. schenckii s.str. In this paper, phylogeny and epidemiology of the members of this clade were investigated for 99 clinical and 36 environmental strains using four genetic loci, viz. rDNA ITS and partial CAL, TEF1, and TEF3; data are compared with amplified fragment length polymorphism (AFLP) genotyping. The four main species of the pathogenic clade were recognised. The species proved to show high degrees of endemicity, which enabled interpretation of literature data where live material or genetic information is lacking. The clade of four species comprised nine subclusters, which often had limited geographic distribution and were separate from each other in all partitions, suggesting low degrees of interbreeding between populations. In contrast, S. globosa exhibited consistent global distribution of identical AFLP types, suggesting another type of dispersal. Sporothrix brasiliensis is known to be involved in an expanding zoonosis and transmitted by cats, whereas S. globosa infections originated from putrid plant material, causing a sapronosis. Sporothrix schenckii s.str., the most variable species within the clade, also had a plant origin, with ecological similarities to that of S. globosa. A hypothesis was put forward that highly specific conditions in the plant material are required to promote the growth of Sporothrix. Fermented, self-heated plant debris may stimulate the thermodependent yeast-like invasive form of the fungus, which facilitates repeated infection of mammals.

New Clonal Strain ofCandida auris, Delhi, India

A new clonal strain of Candida auris is an emerging etiologic agent of fungemia in Delhi, India. In 12 patients in 2 hospitals, it was resistant to fluconazole and genotypically distinct from isolates from South Korea and Japan, as revealed by M13 and amplified fragment length polymorphism typing.

Escherichia coli bacteraemia: 2 years of prospective regional surveillance (2010-12)

To determine the prevalence of antibiotic resistance and the epidemiology of Escherichia coli bacteraemia isolates across the Yorkshire and Humber National Health Service region over 2 years. Ten percent of all E. coli blood culture isolates were collected per month from 14 laboratories across the Yorkshire and Humber region. Individual laboratories submitted antibiotic susceptibility data and isolates were re-tested centrally using the VITEK2(®) system (bioMérieux, France). Isolates were also characterized using PCR to test for the presence of sequences encoding extended-spectrum β-lactamases (ESBLs) and genotyped using amplified fragment length polymorphism (AFLP). Selected isolates were further characterized using multilocus sequence typing. Between July 2010 and June 2012, 770 isolates were examined: 63%, 40%, 14% and 7% of isolates were non-susceptible to ampicillin, trimethoprim, ciprofloxacin and gentamicin, respectively. Eight percent of isolates (n = 63) were ESBL positive; CTX-M group 1 enzymes were the most common (68%). There was a fluctuating trend in the prevalence of resistance to amoxicillin/clavulanic acid (MIC >8 mg/L): July-September 2010, 16%; July-September 2011, 38%; and April-June 2012, 22%. AFLP identified 106 types. The majority of isolates belonged to one of two AFLP types: AFLP 1 [sequence type (ST) 131; 17%] and AFLP 2 (ST73; 18%). ST131 and ST73 were both associated with hospital- and community-onset bacteraemia, and with urinary, hepatobiliary and gastrointestinal sources of infection. ESBL-positive isolates were predominantly ST131 (60%). Continued surveillance of antibiotic resistance among E. coli bacteraemia isolates is necessary to enhance these early baseline data. The variable prevalence of resistance to amoxicillin/clavulanic acid raises concerns, as both E. coli bacteraemia and empirical use of this antibiotic are common.

Prevalence and genotype of Mycoplasma bovis in beef cattle after arrival at a feedlot

To determine the prevalence of Mycoplasma bovis infection in the lungs of cattle at various times after arrival at a feedlot, to measure the relationship between clinical disease status and the concentration and genotype of M bovis within the lungs, and to investigate changes in the genotype of M bovis over time. Bronchoalveolar lavage fluid (BALF) from 328 healthy or pneumonic beef cattle and 20 M bovis isolates obtained from postmortem samples. The concentration of M bovis in BALF was determined via real-time PCR assays, and M bovis isolates from BALF were genotyped via amplified fragment length polymorphism (AFLP) analysis. Prevalence of M bovis in BALF was 1 of 60 (1.7%) at arrival to a feedlot and 26 of 36 (72.2%) and 36 of 42 (85.7%) at ≤ 15 days and 55 days after arrival, respectively. Neither the concentration nor the AFLP type of M bovis in BALF was correlated with clinical disease status. The M bovis AFLP type differed between early and later sampling periods in 14 of 17 cattle. The findings implied spread of M bovis among calves and suggested that host factors and copathogens may determine disease outcomes in infected calves. Chronic pulmonary infection with M bovis may represent a dynamic situation of bacterial clearance and reinfection with strains of different AFLP type, rather than continuous infection with a single clone. These findings impact our understanding of why cattle with chronic pneumonia and polyarthritis syndrome inadequately respond to antimicrobial treatment.

Characterization of imipenem resistance mechanisms in Pseudomonas aeruginosa isolates from Turkey

The emergence of carbapenem resistance in Pseudomonas aeruginosa threatens the efficacy of this important anti-pseudomonal antibiotic class. Between 2003 and 2006, an increase in the number of carbapenem-resistant P. aeruginosa isolates at the Zonguldak Karaelmas University Hospital was observed (Zonguldak, Turkey). To assess the imipenem resistance mechanisms emerging in these P. aeruginosa isolates, they were characterized by amplified fragment length polymorphism typing, which revealed diversity among imipenem-resistant isolates as well as two clonally related outbreak groups. The molecular mechanism of carbapenem resistance was characterized in a representative isolate from each clonal group. Mutational disruption of oprD was the most frequently encountered resistance mechanism (23/27 isolates).

Distribution of coagulase-negative Staphylococcus species from milk and environment of dairy cows differs between herds

In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant pathogens causing intramammary infections (IMI) in dairy cows. The cows’ environment is thought to be a possible source for CNS mastitis and this was investigated in the present paper. A longitudinal field study was carried out in 6 well-managed dairy herds to determine the distribution and epidemiology of various CNS species isolated from milk, causing IMI and living freely in the cows’ environment, respectively. In each herd, quarter milk samples from a cohort of 10 lactating cows and environmental samples from stall air, slatted floor, sawdust from cubicles, and sawdust stock were collected monthly (n=13). Isolates from quarter milk samples (n=134) and the environment (n=637) were identified to species level using amplified fragment length polymorphism (AFLP) genotyping. Staphylococcus chromogenes, S. haemolyticus, S. epidermidis, and S. simulans accounted for 81.3% of all CNS milk isolates. Quarters were considered infected with CNS (positive IMI status) only when 2 out of 3 consecutive milk samples yielded the same CNS AFLP type. The species causing IMI were S. chromogenes (n=35 samples with positive IMI status), S. haemolyticus (n=29), S. simulans (n=14), and S. epidermidis (n=6). The observed persistent IMI cases (n=17) had a mean duration of 149.4 d (range 63.0 to 329.8 d). The CNS species predominating in the environment were S. equorum, S. sciuri, S. haemolyticus, and S. fleurettii. Herd-to-herd differences in distribution of CNS species were observed in both milk and the environment, suggesting that herd-level factors are involved in the establishment of particular species in a dairy herd. Primary reservoirs of the species causing IMI varied. Staphylococcus chromogenes and S. epidermidis were rarely found in the environment, indicating that other reservoirs were more important in their epidemiology. For S. haemolyticus and S. simulans, the environment was found as a reservoir, suggesting that IMI with these species were possibly environmental in origin.

Combined MLST and AFLP typing of Bartonella henselae isolated from cats reveals new sequence types and suggests clonal evolution

Bartonella species are Gram-negative, fastidious bacteria. Bartonella henselae is found in cats and transmitted to humans via cat scratches or bites causing cat-scratch disease, characterized by clinical symptoms with varying severity. The prevalence of bartonellosis among humans in Germany appears to be high, and severe clinical cases have been described. However, epidemiological data of B. henselae in cats are rare. In this study we determined the detection rates of Bartonella ssp. in cats by culture and real-time PCR. Furthermore, B. henselae isolates were genetically characterized by highly discriminatory amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST). Bartonella spp. were isolated by culture from 11 (2.2%) of 507 blood samples. Out of 169 blood samples additionally analyzed by PCR, 28 (16.6%) were found positive for Bartonella spp., illustrating the advantage of PCR in Bartonella spp. detection. PCR-REA identified B. henselae in 27 cats and Bartonella clarridgeiae in one cat. B. henselae isolates from different geographical regions in Germany were genetically characterized by AFLP and MLST. Both methods confirmed genetic diversity of B. henselae on the strain level. MLST identified 11 new sequence types, all of them assigned to three clonal complexes as determined by eBURST. AFLP typing revealed genetic relation among the B. henselae isolates from the same geographical region. Combining AFLP typing and MLST/eBURST analyses revealed that B. henselae of the same AFLP subcluster belonged to the same clonal complex. Altogether these results indicate that B. henselae may evolve clonally.

Carry-over of thermophilic Campylobacter spp. between sequential and adjacent poultry flocks

Nineteen flocks of four poultry species were monitored at a veterinary field station to investigate the distribution and spread of Campylobacter genotypes between sequential and adjacent flocks. Caecal and liver samples were obtained at frequent intervals from birds of all flocks and examined for Campylobacter. Amplified fragment length polymorphism (AFLP) analysis was performed to genotype Campylobacter isolates. Of the 1643 caecal and liver samples investigated, 452 (27.5%) caecal samples and 11 (0.7%) liver samples contained Campylobacter. Of the caecal isolates 76.3% were identified as Campylobacter jejuni and 23.7% were identified as Campylobacter coli. Poultry flocks were largely colonized by more than one AFLP type and an intense exchange of Campylobacter genotypes between different poultry flocks occurred. These findings indicate that multiple genotypes can constitute the Campylobacter population within single poultry flocks, hinting to different sources of exposure and/or genetic drifts within the Campylobacter population. Nevertheless, in most flocks single Campylobacter genotypes predominated. Some strains superseded others resulting in colonization by successive Campylobacter genotypes during the observation period. In conclusion, the data demonstrate that the large genetic diversity of Campylobacter must be considered in epidemiological evaluations and microbial risk assessments of Campylobacter in poultry.

Characterisation of carbapenem-resistant Acinetobacter baumannii outbreak strains producing OXA-58 in Turkey

The aim of this study was to characterise the molecular epidemiology and mechanisms of carbapenem resistance of nosocomial Acinetobacter baumannii isolates in a new university hospital in Turkey. A total of 145 carbapenem-resistant A. baumannii (CRAB) isolates were collected during the period 2003–2006. All isolates were typed by amplified fragment length polymorphism (AFLP) analysis. AFLP analysis showed three predominant clusters consisting of 72, 20 and 12 clinical strains as well as some smaller clusters and 23 unique strains. The three main clonal AFLP types corresponded to three major antibiotic susceptibility patterns. One environmental isolate was found related to the major outbreak clone. The reference type strains of European clones I, II and III were also typed by AFLP and analysed for clonal similarity. Polymerase chain reaction (PCR) analysis of different carbapenem resistance genes showed that strains from each of the three main clusters as well as 79% of the remaining strains harboured the bla OXA-58 gene. No genes encoding the metallo-β-lactamases GIM-1, SIM-1, SPM-1, IMP-like and VIM-like or the oxacillinases OXA-24-like and OXA-23-like were detected. In conclusion, multiple clones of CRAB strains producing OXA-58-type oxacillinase were responsible for a sustained CRAB outbreak occurring in a hospital in Turkey. These isolates were not associated with A. baumannii strains of the major European clones I, II or III.

Demonstration of persistent strains of Campylobacter jejuni within broiler farms over a 1‐year period in Lithuania

The aim of the study was to investigate the flock prevalence of Campylobacter jejuni and Campylobacter coli in broiler farms in Lithuania and to identify possible persistent strains of Camp. jejuni using amplified fragment length polymorphism (AFLP) typing method. During 1 year, 42 broiler flocks from 9 broiler farms were examined to determine the prevalence of Campylobacter-positive broiler flocks in Lithuania. Among 42 broiler flocks examined, 31 flocks (73.8%) were positive for Camp. jejuni and 17 flocks (40.48%) for Camp. coli. Campylobacter jejuni isolates were genotyped by AFLP method using BspDI and BglII restriction enzymes. Typing of 190 isolates generated 50 AFLP genotypes with the highest diversity of strains found in the summer season. Each farm showed one or more predominant AFLP types, and one AFLP type (A32) was found in five broiler farms over a 1-year period. Campylobacter jejuni and Camp. coli are highly prevalent in broiler farms in Lithuania. Farm-specific genotypes were identified in all farms examined. Type A32 was present and persisted in different broiler farms, and a common source of transmission of Camp. jejuni was suspected. For the first time, Camp. jejuni in broiler flocks has been genetically characterized in Lithuania. Persistent strains of Camp. jejuni were detected over one period at the beginning of broiler meat production chain and, therefore, the identification of contamination source of such strains and the mechanism of their particular ability to persist are crucial to establish effective control measures against Camp. jejuni infection in broiler farms.

Amplified Fragment Length Polymorphism and Multi-Locus Sequence Typing for high-resolution genotyping of Listeria monocytogenes from foods and the environment

Standardized tools for typing Listeria monocytogenes isolates are required in epidemiological surveys investigating food-borne disease outbreaks and in the food-processing environment to identify the sources of contamination and routes by which the organisms are spread. In this survey Amplified Fragment Length Polymorphism (AFLP) and Multi-Locus Sequence Typing (MLST) have been used to study 103 L. monocytogenes isolates from food and environmental sources. A total of 62 AFLP types and 66 MLST Sequence Types were identified. AFLP and MLST produced similar results in terms of discriminating power. The Discrimination Index calculated for the two techniques was 0.976 for AFLP and 0.972 for MLST. These values were appreciably higher compared to serotyping (0.739). A good congruence was observed between AFLP and MLST. The present study demonstrated that AFLP and MLST subtyping are suitable tools for studying the epidemiology of L. monocytogenes. The great advantage of MLST over AFLP and other molecular typing methods based on fragment fingerprinting lies in the unambiguity of sequence data while AFLP is less costly and highly processive. In conclusion the two methods can be perfectly integrated for high-resolution genotyping of L. monocytogenes.

Classification of the taxon 2 and taxon 3 complex of Bisgaard within Gallibacterium and description of Gallibacterium melopsittaci sp. nov., Gallibacterium trehalosifermentans sp. nov. and Gallibacterium salpingitidis sp. nov.

This investigation was based on 23 isolates from several European countries collected over the past 30 years, and included characterization of all isolates. Published data on amplified fragment length polymorphism typing of isolates representing all biovars as well as protein profiles were used to select strains that were then further characterized by polyamine profiling and sequencing of 16S rRNA, infB, rpoB and recN genes. Comparison of 16S rRNA gene sequences revealed a monophyletic group within the avian 16S rRNA group of the Pasteurellaceae, which currently includes the genera Avibacterium, Gallibacterium and Volucribacter. Five monophyletic subgroups related to Gallibacterium anatis were recognized by 16S rRNA, rpoB, infB and recN gene sequence comparisons. Whole-genome similarity between strains of the five subgroups and the type strain of G. anatis calculated from recN sequences allowed us to classify them within the genus Gallibacterium. In addition, phenotypic data including biochemical traits, protein profiling and polyamine patterns clearly indicated that these taxa are related. Major phenotypic diversity was observed for 16S rRNA gene sequence groups. Furthermore, comparison of whole-genome similarities, phenotypic data and published data on amplified fragment length polymorphism and protein profiling revealed that each of the five groups present unique properties that allow the proposal of three novel species of Gallibacterium, for which we propose the names Gallibacterium melopsittaci sp. nov. (type strain F450(T) =CCUG 36331(T) =CCM 7538(T)), Gallibacterium trehalosifermentans sp. nov. (type strain 52/S3/90(T) =CCUG 55631(T) =CCM 7539(T)) and Gallibacterium salpingitidis sp. nov. (type strain F150(T) =CCUG 15564(T) =CCUG 36325(T) =NCTC 11414(T)), a novel genomospecies 3 of Gallibacterium and an unnamed taxon (group V). An emended description of the genus Gallibacterium is also presented.

Drug-resistant gene based genotyping for Acinetobacter baumannii in tracing epidemiological events and for clinical treatment within nosocomial settings

Background Acinetobacter baumannii has emerged as an important pathogen related to serious infections and nosocomial outbreaks around the world. However, of the frequently used methods, pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) in Acinetobacter baumannii genotyping lack the direct molecular proof of drug resistance. This study was conducted to establish a typing method based on drug resistant gene identification in contrast to traditional PFGE and AFLP in the period of nosocomial epidemic or outbreak. Methods From January 2005 to October 2005, twenty-seven strains of Acinetobacter species from Intensive Care Units, the Second Affiliated Hospital in Ningbo were isolated, including both epidemic and sporadic events. Susceptibility test, PFGE, AFLP and drug resistance gene typing (DRGT) were carried out to confirm the drug resistance and analyze the genotyping, respectively. PFGE was used as a reference to evaluate the typeability of DRGT and AFLP. Results Twenty-seven strains of Acinetobacter displayed multiple antibiotic resistance and drug resistant genes, and β-lactamase genes were detected in 85.2% strains. The result of DRGT was comparable to PFGE in Acinetobacter strains with different drug resistance though a little difference existed, and even suggested a molecular evolution course of different drug-resistant strains. AFLP showed great polymorphism between strains and had weak ability in distinguishing the drug resistance. Conclusion Compared to AFLP and PFGE, DRGT is useful to analyze localized molecular epidemiology of nosocomial infections and outbreaks, which would benefit clinical diagnosis and therapy.

Molecular epidemiological investigation of an outbreak of Pseudomonas aeruginosa infection in an SCT unit

From May to October 2006, six severe Pseudomonas aeruginosa infections were diagnosed in patients undergoing SCT in the SCT unit of the Careggi hospital (Florence, Italy). Four of the infected patients were treated consecutively in the same room (room N). On the hypothesis of a possible environmental source of infection, samples were collected from different sites that had potential for cross-contamination throughout the SCT unit, including the electrolytic chloroxidant disinfectant used for hand washing (Irgasan) and the disinfectant used for facilities cleaning. Four of the environmental samples were positive for P. aeruginosa: three Irgansan soap samples and a tap swab sample from the staff cleaning and dressing room. The AFLP (amplified fragment length polymorphism) typing method employed to evaluate strain clonality showed that the isolates from the patients who had shared the same room and an isolate from Irgasan soap had a significant molecular similarity (dice index higher than 0.93). After adequate control measures, no subsequent environmental sample proved positive for P. aeruginosa. These data strongly support the hypothesis of the clonal origin of the infective strains and suggest an environmental source of infection. The AFLP method was fast enough to allow a 'real-time' monitoring of the outbreak, permitting additional preventive measures.