MLVA Research Articles

Phenotypic and genotypic characteristics of Brucella isolates from the Republic of Kazakhstan.

The purposes of this study were to determine phenotypic and genotypic characteristics of Brucella isolates from the Republic of Kazakhstan and to determine their biotype. The focus was laid on culture-morphological, biochemical, and biological properties of 59 Brucella isolates from primary cultures. Material was isolated from blood and tissue of serum-positive killed, dead diseased, or aborted domestic cattle from different regions of Kazakhstan where brucellosis is a common problem. Multiple-locus variable number tandem repeat analysis (MLVA) of all strains, isolated in different regions, has shown that Brucella isolates from the epizootic form two clusters. Based on the comparison with strains available in the MLVA database, B. abortus 0015/B is alike the B. abortus strains isolated from Italy and Portugal. B. melitensis 0016/B isolated from the Almaty region fits the third cluster and is alike the B. melitensis strains isolated from humans in Turkey, China, and Portugal. More than 90% of the overall B. abortus samples were isolated from the northern regions of the East and West Kazakhstan, while B. melitensis strains were registered in the southeast Kazakhstan. The most frequently recorded B. abortus biovar is biovar 3. The most frequently recorded B. melitensis biovars are biovars 1 and 3. SIGNIFICANCE AND IMPACT OF STUDY: These results contribute to a better understanding of the geographic pattern of Brucella infection in Kazakh cattle also important for developing the specific control measures. The results of current research can be used for creating a gene bank of Brucella strains circulating in Kazakhstan for producing diagnostic and therapeutic agents. The research material will be used to solve the problems of genetic characterization of Brucella species and to establish the phylogenetic relationships of strains.

Mycoplasma hyopneumoniae variability: Current trends and proposed terminology for genomic classification.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the aetiologic agent of enzootic pneumonia in swine, a prevalent chronic respiratory disease worldwide. Mycoplasma hyopneumoniae is a small, self-replicating microorganism that possesses several characteristics allowing for limited biosynthetic abilities, resulting in the fastidious, host-specific growth and unique pathogenic properties of this microorganism. Variation across several isolates of M. hyopneumoniae has been described at antigenic, proteomic, transcriptomic, pathogenic and genomic levels. The microorganism possesses a minimal number of genes that regulate the transcription process. Post-translational modifications (PTM) occur frequently in a wide range of functional proteins. The PTM by which M. hyopneumoniae regulates its surface topography could play key roles in cell adhesion, evasion and/or modulation of the host immune system. The clinical outcome of M. hyopneumoniae infections is determined by different factors, such as housing conditions, management practices, co-infections and also by virulence differences among M. hyopneumoniae isolates. Factors contributing to adherence and colonization as well as the capacity to modulate inflammatory and immune responses might be crucial. Different components of the cell membrane (i.e. proteins, glycoproteins and lipoproteins) may serve as adhesins and/or be toxic for the respiratory tract cells. Mechanisms leading to virulence are complex and more research is needed to identify markers for virulence. The utilization of typing methods and complete or partial-gene sequencing for M. hyopneumoniae characterization has increased in diagnostic laboratories as control and elimination strategies for this microorganism are attempted worldwide. A commonly employed molecular typing method for M. hyopneumoniae is Multiple-Locus Variable number tandem repeat Analysis (MLVA). The agreement of a shared terminology and classification for the various techniques, specifically MLVA, has not been described, which makes inferences across the literature unsuitable. Therefore, molecular trends for M. hyopneumoniae have been outlined and a common terminology and classification based on Variable Number Tandem Repeats (VNTR) types has been proposed.

OPTIMIZATION OF MULTIPLE-LOCUS VARIABLE-NUMBER TANDEM-REPEAT ANALYSIS FOR BACILLUS ANTRACIS GENOTYPING

Bacillus anthracis is the causative agent of anthrax, a highly dangerous human and veterinary disease. B. anthracis has high pathogenicity, resulting in a high mortality rate, with possible long-term preservation of the live pathogen in environmental conditions. Thus, genotyping of circulating B. anthracis strains is an integral part of epidemiological surveillance worldwide. Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a highly discriminatory method for genotyping bacterial species with a conserved genome such as B. anthracis. Although there is already a well-established MLVA typing scheme, protocol optimization and verification of the results are required in certain cases. This paper presents the results of optimization of the MLVA-31 protocol, which was verified in four B. anthracis strains that were isolated in Kazakhstan. The actual VNTR repeat sizes differed from those obtained by capillary electrophoresis at all VNTR loci. Moreover, absence of B. anthracis reference strains made allele identification difficult for 90% of the loci. In six loci, the actual sizes differed by one or more VNTRs from the sizes defined by capillary electrophoresis. These results indicate that availability of B. anthracis reference strains will allow for verification of genotyping results, regardless of the particular reagents and equipment used for capillary electrophoresis, thus enabling more efficient epidemiological monitoring at the local and global levels.

An international outbreak of Salmonella enterica serotype Enteritidis linked to eggs from Poland: a microbiological and epidemiological study

Salmonella spp are a major cause of food-borne outbreaks in Europe. We investigated a large multi-country outbreak of Salmonella enterica serotype Enteritidis in the EU and European Economic Area (EEA). A confirmed case was defined as a laboratory-confirmed infection with the outbreak strains of S Enteritidis based on whole-genome sequencing (WGS), occurring between May 1, 2015, and Oct 31, 2018. A probable case was defined as laboratory-confirmed infection with S Enteritidis with the multiple-locus variable-number tandem repeat analysis outbreak profile. Multi-country epidemiological, trace-back, trace-forward, and environmental investigations were done. We did a case-control study including confirmed and probable cases and controls randomly sampled from the population registry (frequency matched by age, sex, and postal code). Odds ratios (ORs) for exposure rates between cases and controls were calculated with unmatched univariable and multivariable logistic regression. 18 EU and EEA countries reported 838 confirmed and 371 probable cases. 509 (42%) cases were reported in 2016, after which the number of cases steadily increased. The case-control study results showed that cases more often ate in food establishments than did controls (OR 3·4 [95% CI 1·6-7·3]), but no specific food item was identified. Recipe-based food trace-back investigations among cases who ate in food establishments identified eggs from Poland as the vehicle of infection in October, 2016. Phylogenetic analysis identified two strains of S Enteritidis in human cases that were subsequently identified in salmonella-positive eggs and primary production premises in Poland, confirming the source of the outbreak. After control measures were implemented, the number of cases decreased, but increased again in March, 2017, and the increase continued into 2018. This outbreak highlights the public health value of multi-country sharing of epidemiological, trace-back, and microbiological data. The re-emergence of cases suggests that outbreak strains have continued to enter the food chain, although changes in strain population dynamics and fewer cases indicate that control measures had some effect. Routine use of WGS in salmonella surveillance and outbreak response promises to identify and stop outbreaks in the future. European Centre for Disease Prevention and Control; Directorate General for Health and Food Safety, European Commission; and National Public Health and Food Safety Institutes of the authors' countries (see Acknowledgments for full list).

Molecular and clinical characteristics of severe Mycoplasma pneumoniae pneumonia in children.

To analyze the molecular and the clinical characteristics of Mycoplasma pneumoniae (Mp) pneumonia (MPP) and to explore the related factors predicting severe MPP (SMPP). A total of 423 pediatric cases of MPP were retrospectively analyzed, in 2013-2017, in Beijing, China. Clinical information was collected from the medical records. Mp-positive specimens were characterized using P1 typing and multiple locus variable-number tandem repeat analysis (MLVA). The macrolide resistance-associated mutations were also detected. The predominant genotype was P1-1 (88.2%) and M4-5-7-2 (87.5%), whereas percentages of type P1-2 and M3-5-6-2 increased across the 5-year period. The mutation rate of genotype M4-5-7-2 (365/370, 98.6%) was significantly higher than that of the genotype M3-5-6-2 (15/48, 32.25%; P = 0.000). Overall, 180 (42.6%) of the 423 Mp-positive patients were coinfected with other pathogens. Respiratory syncytial virus coinfection (24/180, 13.3%) was more common in cases typed M3-5-6-2 (4/23, 17.4%) than that of M4-5-7-2 (20/155, 12.9%; P = 0.038). Pleural effusion accounted for 52.6% (169/321) of the observed complications. In the mono-infection cases, cases typed M3-5-6-2 (56%, 14/25) were significantly (P = 0.020) associated with pleural effusion compared with those typed M4-5-7-2 (32.6%, 70/215); 84% (21/25) of specimens typed M3-5-6-2 were diagnosed as SMPP, whereas 63.7% (137/215) of specimens typed M4-5-7-2 were diagnosed as SMPP (P = 0.043). In our study, we proposed for the first time that the mono-infection patients with Mp typed M3-5-6-2 appear to have a higher risk for progressing to SMPP. MLVA typing can provide hints on the clinical characteristics of Mpp.

Molecular characterization of Brucella species from Zimbabwe.

Brucella abortus and B. melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B. ovis, B. abortus, B. canis and B. suis, with B. canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B. suis and B. abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world.

Genotypic methods for epidemiological typing of Staphylococcus aureus

Staphylococcus aureus is an important pathogen causing both hospital and community-acquired infections. S. aureus strains are often resistant to commonly used antibiotics, thus development of techniques to rapidly identify and genotype is essential in order to control the dissemination of multidrug resistant strains of S. aureus in the environment, which could lead to an epidemic. Typing methods can be divided into those based on phenotypic and those based on genetic characteristics of the examined microorganism. The most reliable genotypic methods in S. aureus epidemiological studies were described. These would include the following: Plasmid Profile Analysis (PPA), Pulsed-Field Gel Electrophoresis (PFGE), Multi-Locus Sequence Typing (MLST), Spa Typing, Random Amplified Polymorphic DNA (RAPD), Amplified-Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP), Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and Whole Genome Sequencing (WGS). All of these methods have been compared in terms of their reproducibility, costs, time of analysis, differentiation potential and exemplary applications in epidemiological studies of S. aureus. Despite there being a variety of methods currently used for genotyping, not all of them meet the criteria set by different kinds of epidemiological investigations. Because of the large amount of generated data and decrease of costs, the most popular methods used for S. aureus genotyping are based on sequencing techniques.

Bioburden and transmission of pathogenic bacteria through elevator channel during endoscopic retrograde cholangiopancreatography: application of multiple-locus variable-number tandem-repeat analysis for characterization of clonal strains

ABSTRACTObjectives: Endogenous and exogenous infection of the biliary tract could occur during endoscopic retrograde cholangiopancreatography.Methods: Bile samples of patients with hepatobiliary diseases, and swab samples of elevator channel samples of duodenoscope and washing instruments were prepared simultaneously and cultured aerobically and anaerobically. Antimicrobial susceptibility of the most common characterized bacterial species was tested, and their genetic relatedness was analyzed by multiple locus variable number of tandem repeats method.Results: Contamination with Pseudomonas aeruginosa was detected in 38.2% of the elevator channels’ and 26.6% of the bile samples. Staphylococcus aureus, Enterococcus spp., Staphylococcus epidermidis, Escherichia coli, Enterobacter spp., and Clostridium perfringenes were among other bacterial isolates in the elevator channel swab samples. Highest antimicrobial resistance rate among P. aeruginosa isolates from the bile and swab samples were detected against gentamicin (100% and 73%, respectively), while the lowest one was measured to piperacillin-tazobactam (25% and 0%, respectively). Out of the 27 distinct MLVA profiles, relatedness of P. aeruginosa strains in the bile samples compared with those from the elevators was shown in three series of the samples.Conclusion: Identity of P. aeruginosa strains among the bile and elevator channel samples showed possibility of cross-contamination among patients even at distinct time intervals.Expert opinion: Bacterial infection is considered as main complications of ERCP. Entry of bacteria into the biliary tract via contaminated device and its related instruments and their proliferation in this tissue could cause serious infections. To prevent this side effect, reprocessing of medical equipment via standard cleaning and disinfection procedures are needed. Our results showed incompliance of methods used for endoscope cleaning and disinfection procedure. Although host risk factors, such as sphincterotomy, could increase rate of infection with different types of bacteria, their ability for formation of biofilm and spore, which could help them to resist disinfectants and washing procedures seems to be main cause of persistent colonization and transmission among different patients. New standards for disinfection compared with currently used methods and use of materials to eliminate formation of bacterial microcolonies seem to be necessary to prevent cross-contamination.

Antimicrobial agent susceptibilities of Legionella pneumophila MLVA-8 genotypes

Legionella pneumophila causes human lung infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA-8). Legionella infections in humans occur as a result of inhalation of bacteria-containing aerosols, thus, our aim was to study the antimicrobial susceptibilities of different MLVA-8 genotypes to ten commonly used antimicrobial agents in legionellosis therapy. Epidemiological cut-off values were determined for all antibiotics. Significant differences were found between the antimicrobial agents’ susceptibilities of the three studied environmental genotypes (Gt4, Gt6, and Gt15). Each genotype exhibited a significantly different susceptibility profile, with Gt4 strains (Sequence Type 1) significantly more resistant towards most studied antimicrobial agents. In contrast, Gt6 strains (also Sequence Type 1) were more susceptible to six of the ten studied antimicrobial agents compared to the other genotypes. Our findings show that environmental strains isolated from adjacent points of the same water system, exhibit distinct antimicrobial resistance profiles. These differences highlight the importance of susceptibility testing of Legionella strains. In Israel, the most extensively used macrolide for pneumonia is azithromycin. Our results point at the fact that clarithromycin (another macrolide) and trimethoprim with sulfamethoxazole (SXT) were the most effective antimicrobial agents towards L. pneumophila strains. Moreover, legionellosis can be caused by multiple L. pneumophila genotypes, thus, the treatment approach should be the use of combined antibiotic therapy. Further studies are needed to evaluate specific antimicrobial combinations for legionellosis therapy.

Network properties of salmonella epidemics

We examine non-typhoidal Salmonella (S. Typhimurium or STM) epidemics as complex systems, driven by evolution and interactions of diverse microbial strains, and focus on emergence of successful strains. Our findings challenge the established view that seasonal epidemics are associated with random sets of co-circulating STM genotypes. We use high-resolution molecular genotyping data comprising 17,107 STM isolates representing nine consecutive seasonal epidemics in Australia, genotyped by multiple-locus variable-number tandem-repeats analysis (MLVA). From these data, we infer weighted undirected networks based on distances between the MLVA profiles, depicting epidemics as networks of individual bacterial strains. The network analysis demonstrated dichotomy in STM populations which split into two distinct genetic branches, with markedly different prevalences. This distinction revealed the emergence of dominant STM strains defined by their local network topological properties, such as centrality, while correlating the development of new epidemics with global network features, such as small-world propensity.

First report of Salmonella 1,4,[5],12:i:- in free-ranging striped dolphins (Stenella coeruleoalba), Italy

Between 2015 and the beginning of 2018 (January-March), 30 cetaceans were found stranded along the Ligurian Sea coast of Italy. Necropsies were performed in 22 cases and infectious diseases resulted the most common cause of death. Three striped dolphins, showed a severe coinfection involving the monophasic variant of Salmonella Typhimurium (Salmonella 1,4,[5],12:i:-). The isolates were characterized based on antimicrobial resistance, Multiple-Locus Variable-number tandem-repeat Analysis (MLVA) and whole-genome sequencing (WGS). All isolates demonstrated the same multidrug resistant genotype (ASSuT isolates), showed three different MLVA profiles, two of which closely related, and were identified as Sequence Type 34. Moreover, Single nucleotide polymorphisms (SNP) analysis confirmed strong correlations between two out of the three isolates. To our knowledge, S. 1,4,[5],12:i:-, one of the most common serovars in cases of human infection and food sources worldwide, has not previously been described in marine mammals, and reports of Salmonella-associated disease in free-ranging cetaceans are rare. These results highlight the role of cetaceans as sentinel species for zoonotic and terrestrial pathogens in the marine environment, suggest a potential risk for cetaceans and public health along the North Western Italian coastline and indicate cetaceans as a novel potential reservoir for one of the most widespread Salmonella serovars.

Evaluation of random amplified polymorphic DNA and multiple-locus variable number tandem repeat analyses for Mycoplasma cynos

Mycoplasma spp. can cause diseases of the respiratory system as well as urogenital infections, infertility, and anemia. The members of this genus have a low G + C content compared to other bacteria. Because primers used in the random amplified polymorphic DNA (RAPD) technique are only 10 bp long and have high GC content, this method can be inadequate for genotyping Mycoplasma spp. isolates. The aim of this study was to develop and evaluate multiple-locus variable number tandem repeat analysis (MLVA) and two-primer RAPD (TP-RAPD) procedures for subtyping Mycoplasma cynos isolates.A total of 55 M. cynos isolates obtained from 162 bronchoalveolar lavage fluid samples from shelter and pet dogs were used in this study. Seventy-four tandem repeat regions were detected in the M. cynos genome, and two of these loci were determined to be suitable and used for development of the MLVA scheme. The results of variable number tandem repeat (VNTR) analysis and TP-RAPD-PCR were compared with RAPD-PCR. The discriminatory power of TP-RAPD-PCR (Hunter-Gaston diversity index [HGDI] = 0.84) was higher than those of RAPD-PCR (HGDI = 0.727), VNTR1 (HGDI = 0.8), and VNTR3 (HGDI = 0.757). We observed that the TP-RAPD-PCR and MLVA methods provide clearer data and are more successful in determining genetic diversity, in contrast to the RAPD-PCR method for this species.

Molecular epidemiology of Leptospira interrogans in Rattus norvegicus in Hanoi, Vietnam

Leptospirosis is a zoonotic disease that is caused by pathogenic spirochaetes of Leptospira spp. and it has become a public health concern in urban localities in the tropics. Rats are important reservoir animals for the transmission of leptospirosis in urban areas. Leptospirosis is considered endemic in Vietnam. However, information on the causative Leptospira genotypes and serotypes in the country is limited. We investigated the carrier status of Leptospira spp. in rats captured in Hanoi by culturing and DNA detection. Isolates were characterized using a serological method and multiple-locus variable-number tandem repeat analysis (MLVA).We captured 144 rats (1 Rattus argentiventer, 135 R. norvegicus, and 8 R. rattus) and obtained 17 L. interrogans, determined by rrs sequencing, from R. norvegicus (12.6%). Sixteen of the isolates were serogroup Bataviae. Five of the 16 isolates exhibited an MLVA type identical to that of the serovar Bataviae reference strain Van Tienen, while there were nine repeats for the other 11 isolates at VNTR31 compared with the reference strain. The remaining isolate grew poorly, and we were unable to determine its serogroup. However, it had an MLVA type matching those of serogroup Pomona strains isolated from R. norvegicus in Japan. Three different flaB sequences were detected in 23 out of 81 R. norvegicus kidney tissue samples (28.4%) using nested PCR followed by DNA sequencing. Two of the sequences were identical with those of serogroups Bataviae and Pomona, and no strain with another sequence was detected in the present study.The present study reveals a high prevalence rate of L. interrogans among R. norvegicus in Hanoi, Vietnam, indicating a potential risk of rat-borne leptospirosis in the area. The present study also demonstrates that a fastidious L. interrogans strain circulates among rats and that molecular detection is crucial in facilitating the accurate determination of reservoir animals.

Genotypic identification of Pseudomonas aeruginosa strains isolated from patients with urinary tract infection

BackgroundNumerous nosocomial infections including urinary tract infection (UTI) have been reported to be linked to Pseudomonas aeruginosa (P. aeruginosa). This bacterium is one of the most common pathogen colonized in the urinary tract. The main purpose of this study was to evaluated the presence of antibiotic resistance genes and also the most frequent genotype patterns of P. aeruginosa in the patients with UTI hospitalized in different wards of hospitals. Materials and methodsIn this study, 70 strains of P. aeruginosa isolated of urine samples from the patients with UTI were assessed. The isolated strains were genotyped using Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) method. We have also analyzed the presence of TEM and SHV resistant genes in the isolates. ResultsA total of 70 P. aeruginosa strains was isolated from the UTI patients. Based on MLVA method, 61 various genotypes of P. aeruginosa were identified which grouped into two main clusters and 4 sub-clusters. Moreover, approximately 80% and 70% of isolated strains carried the TEM and SHV resistance genes, respectively. ConclusionOur findings showed that the majority of patients hospitalized in different wards of hospitals have experienced the urinary tract infection caused by P. aeruginosa. According to the genotyping results, a high diversity of the P. aeruginosa population was observed in the patients with UTI. Our results can provide a better understanding of the P. aeruginosa genotype distribution and epidemiology of infection, which can be applied as basic data for future antibiotic therapies.

Polyclonal spread of multiple genotypes of Mycoplasma pneumoniae in semi-closed settings in Yamagata, Japan.

To clarify the spread of Mycoplasma pneumoniae infections in semi-closed settings such as schools and family homes using molecular typing methods. We retrospectively searched for school- and family-based clusters of M. pneumoniae infections based on information regarding patients from whom M. pneumoniae strains had been isolated between 2011 and 2013 in Yamagata, Japan. The molecular typing profile, including the P1 type and the four-locus (Mpn13, 14, 15 and 16) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) type, was obtained from our previous study. We identified 11 school-based clusters involving 71 patients and 16 family-based clusters involving 38 patients, including 14 duplications between these types of clusters. A total of 95M. pneumoniae strains isolated from those patients were divided into 4 genotypes: 33 strains of type 4-5-7-2, 1; 31 of type 4-5-7-3, 1; 24 of type 3-5-6-2, 2c; and 7 of type 3-5-6-2, 2a. Of the 11 school-based clusters, 6 clusters (54.5%) consisted of multiple genotypes, and the remaining 5 clusters consisted of a single genotype. Moreover, the presence of multiple genotypes was identified in three classrooms of a school. On the other hand, in 14 (87.5%) of the 16 family-based clusters, the genotypes of the M. pneumoniae strains isolated from each family member were identical. The spread of M. pneumoniae infection in schools is likely polyclonal, since M. pneumoniae strains are brought into schools from various sites, such as family homes, which are important sites of disease transmission.

Distribution of Mycobacterium leprae genotypes from Surabaya and Bandung Clinical Isolates by Multiple Locus Variable Number of Tandem Repeat Analysis

Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis has been proposed as a means of genotyping for tracking leprosy transmission due to tandem repeats’ potential as genetic markers to differentiate M. leprae strains. However, characteristics of polymorphism can vary depending on the population. This study aimed to compare the copy number of repeats in four genetic markers: TTC, AC8a, AC9 and 6-7 in leprosy patients from Surabaya and Bandung. Twenty three patients from Dr. Soetomo General Hospital and 21 from Hasan Sadikin Hospital were recruited. Multiple locus VNTR analysis was applied using total DNA extracts from Slit Skin Smear (SSS). From Surabaya, 7 samples showed the same copy number of four genetic markers (TTC=15; AC8a=10; AC9=10 and 6-7=6) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6); as for samples from Bandung, 2 showed the same copy number (TTC=15; AC8a=8; AC9=10 and 6-7=8) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6). The multiple locus VNTR analysis showed two identical M. leprae VNTR profiles from Bandung and Surabaya which supports the use of VNTR loci for transmission studies.

First Description of Macrolide-Resistant Mycoplasma pneumoniae in Adults with Community-Acquired Pneumonia in Italy.

Background Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP). This cross-sectional study aimed to determine the prevalence of macrolide-resistant M. pneumoniae strains in a convenience series of 234 adult hospitalised and nonhospitalised subjects with a diagnosis of CAP in January 2013 to April 2015 in South Italy. Methods Respiratory samples were subjected to real-time PCR. In M. pneumoniae-positive samples, domain V of 23S rRNA was sequenced to detect resistance-conferring point mutations. P1 major adhesion protein typing and multiple loci variable-number tandem repeat analysis (MLVA) were also performed. Results Of the 234 samples, 15 (6.4%) were positive for M. pneumoniae. Three of these had a macrolide-resistant genotype: two and one had A2063G and A2064G mutations, respectively. Fourteen of the 15 strains were subtyped: half had subtype 1 and half had subtype 2. Eight strains underwent MLVA profiling: one each had the J, A, and Z type. The remainder was unclassifiable. Conclusions This novel discovery of macrolide-resistant M. pneumoniae strains in adults with CAP in Italy suggests that there may be increasing circulation of these strains in the population. To facilitate rapid optimization of the antibiotic strategy in Italy, macrolide resistance should be monitored by a surveillance system that is based on molecular methods.

Hospital outbreak due to Clostridium difficile ribotype 018 (RT018) in Southern Germany

Clostridium (Clostridioides) difficile is the main cause of nosocomial diarrhoea. Ribotype 018 (RT018) has been recognized as the predominant strain responsible for C. difficile infection (CDI) in Italy, whereas in most other European countries only sporadic RT018 cases occur.Between August and October 2015, a suspected C. difficile outbreak at two associated hospitals in Southern Germany was investigated by comprehensive molecular typing. Surprisingly, RT018 was detected in 9/82 CDI patients, which has never been described before in a German outbreak. Phenotypic analysis revealed fluoroquinolone and macrolide resistance. Genetic subtyping using multiple-locus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS) was performed and outbreak isolates were directly compared to sporadic German RT018 isolates and to epidemic ones from Milan, Northern Italy. Molecular typing confirmed a hospital outbreak with closely related RT018 isolates. Both, MLVA and WGS revealed high similarity of outbreak strains with epidemic isolates from Italy, but low similarity to other German isolates. Comparison between both typing strategies showed that ribotyping in combination with MLVA was appropriate to identify related isolates and clonal complexes, whereas WGS provided a better discrimination with more detailed information about the phylogenetic relationship of isolates. This is the first hospital outbreak in Germany presumably caused by cross-national transmission of an Italian epidemic RT018 strain.

Improvement of Multiple-Locus VNTR Analysis Typing Scheme for Helicobacter pylori

Aims: To improve a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay for Helicobacter pylori typing.
 Materials and Methods: Polymorphic VNTRs were searched by Gene Expert. The distribution and polymorphism of each VNTR locus were analyzed in 18 H. pylori genomes from the NCBI genome database by BLAST and were compared with a collection of 15 clinical H. pylori strains. The MLVA assay was compared with MLST-typing for discriminating H. pylori isolates.
 Results: Twelve VNTR loci were identified by bioinformatic screening of H. pylori genomes, and five of them were highly polymorphic. Therefore, an MLVA assay composed of five VNTR loci was developed with greatest discriminatory power.
 Conclusion: MLVA typing is a faster and more standardized method for studying the genetic relatedness of H. pylori isolates. At preliminary stage it is sufficient to use only 3 VNTR loci for the differentiation of H. pylori strains.

Genetic diversity of Mycoplasma hyopneumoniae in Mendoza province

In Argentina, enzootic pneumonia (EP) is highly prevalent and different genetic types of Mycoplasma hyopneumoniae have been identified. However, there is a lack of information about prevalence and other epidemiological aspects of EP in Mendoza province. A multiple Locus variable-number tandem repeat analysis (MLVA) targeting P97 R1, P97 R1A and P146 R3 loci was used to assess the genetic diversity of M. hyopneumoniae from clinical specimens recovered from pigs from five herds located in different districts of Mendoza province. M. hyopneumoniae could be typed from 27 bronchoalveolar lavages (BAL) specimens, and eight different MLVA types were identified. This is the first report about diversity of M. hyopneumoniae in Mendoza. Results obtained in this work allow drawing a better picture of the genetic diversity of this pathogen in Argentina.