Multilocus Variable Tandem Repeat Analysis Research Articles

Investigation of virulence factors, phylogenetic grouping, multiple-locus variable number tandem repeat analysis, and antimicrobial susceptibility of E. coli isolated from aborted bovine fetal tissue.

Escherichia coli is an important microorganism for cattle breeding. The aim of this study was to investigate the presence of phylogenetic groups, virulence factors, genotyping with multi-locus variable tandem repeat analysis (MLVA), and susceptibility to commonly used antimicrobial agents in E. coli strains isolated from aborted bovine fetal samples. In this study, phylogrouping and various virulence genes were analyzed by PCR in E. coli strains isolated from 637 bovine fetal tissue samples. Consequently, E. coli was isolated and identified in 24 samples in culture. Of the 24 isolates identified as positive, 12.5% were defined as group A, 83.3% as B1, and 4.2% as group B2. Of the E. coli isolates, virulence factor fimH was identified in eight (33.3%), traT in 15 (62.5%), ompT in five (20.8%), CNF1 in one (4.16%), and CNF2 in six (25%). Seven genotypic groups were determined as a result of the analysis with the MLVA 10 method. According to the antimicrobial susceptibility test results, high resistance was determined against amoxicillin/clavulanic acid and oxytetracycline. In conclusion, strains of E. coli containing CNF1, CNF2, fimH, traT, and ompT virulence factors can be associated with bovine abortions. It is noteworthy that the dominant phylogenetic group B1 has been observed in cases of cattle abortions.

Clostridioides difficile infections were predominantly driven by fluoroquinolone-resistant Clostridioides difficile ribotypes 176 and 001 in Slovakia in 2018–2019

To investigate the epidemiology of Clostridioides difficile infection (CDI) in Slovakian hospitals after the emergence of ribotype 176 (027-like) in 2016. Between 2018 and 2019, European Centre for Disease Control and Prevention CDI surveillance protocol v2.3 was applied to 14 hospitals, with additional data collected on recent antimicrobial use and the characterization of C. difficile isolates. The mean hospital incidence of CDI was 4.1 cases per 10,000 patient bed-days. One hundred and five (27.6%) in-hospital deaths were reported among the 381 cases. Antimicrobial treatment within the previous 4 weeks was recorded in 90.5% (333/368) of cases. Ribotype (RT)176 was detected in 50% (n=185/370, 14 hospitals) and RT001 was detected in 34.6% (n=128/370,13/14 hospitals) of cases with RT data. Overall, 86% (n=318/370) of isolates were resistant to moxifloxacin by Thr82Ile in GyrA (99.7%). Multi-locus variable tandem repeat analysis showed clonal relatedness of predominant RTs within and between hospitals. Seven of 14 sequenced RT176 isolates and five of 13 RT001 isolates showed between zero and three allelic differences by whole-genome multi-locus sequence typing. The majority of sequenced isolates (24/27) carried the erm(B) gene and 16/27 also carried the aac(6')-aph(2'') gene with the corresponding antimicrobial susceptibility phenotypes. Nine RT176 strains carried the cfr(E)gene and one RT001 strain carried the cfr(C) gene, but without linezolid resistance. The newly-predominant RT176 and endemic RT001 are driving the epidemiology of CDI in Slovakia. In addition to fluoroquinolones, the use of macrolide-lincosamide-streptogramin B antibiotics can represent another driving force for the spread of these epidemic lineages. In C. difficile, linezolid resistance should be confirmed phenotypically in strains with detected cfr gene(s).

Founder-flush effect found in Vibrio cholerae by MLVA of Nigerian samples

Introduction: We analyzed the multilocus variable tandem repeat analysis (MLVA) genotypes from DNA of toxigenic Vibrio cholerae O1 patient isolates from Nigeria collected by the Nigerian Institute of Medical Research between 2016 to 2021. Findings: Sixty distinct MLVA genotypes were observed among the 144 Isolates. There were two large clonal complexes (CC) of related genotypes that differed by change at a single locus. The locus with the most alleles was P5. Each genotype is represented by the number of repeats at each of the five loci, in order. The first CC was observed from 2016 to 2019, but not afterwards. A single genotype of the first CC was observed in 2016, 5 genotypes in 2017, 6 in 2018 including 2 previously observed in 2017 and 1 in 2019. The second CC was observed only in 2021. Three genotypes - 8,4,6,18,y where y = 33, 34 or 35 – were observed in isolates collected at multiple locations. These genotypes were observed in 8, 7 and 7 of the 11 outbreaks, respectively. All three genotypes were present in Gombe, Plateau and Jigawa. Only one outbreak did not have any of these three genotypes. 15 of 18 other genotypes were unique to a location. In Gombe, Katsina, Kebbi, Lagos, Niger, Plateau and Taraba, an isolate with a founder genotype (8,4,6,18,y) was collected prior to isolates with genotypes unique to that site. Discussion: The genotypes of V. cholerae isolates collected from the outbreaks of cholera follow a founder-flush pattern. One or more genotype(s) [the founders] give(s) rise to multiple other genotypes as the outbreak expands and spreads [the flush] over time and space in Nigeria. Our data and analyses are consistent with the founder-flush phenomena. Keywords: cholera, MLVA, founder-flush, Nigeria, surveillance, Africa, Vibrio cholerae

Multiple-locus variable-number tandem repeat analysis for genotyping of erythromycin-resistant group B streptococci in Iran

BackgroundGroup B Streptococcus (GBS or S. agalactiae) is an important pathogen causing severe invasive diseases in neonates, pregnant women, and adults with underlying medical conditions. MethodsTo investigate the incidence of resistance to macrolide, lincosamide and streptogramin type B (MLSB) antibiotics, macrolide and tetracycline resistance determinants and genetic relationships, a total of 146 clinical isolates of GBS were collected from Tehran, Iran. The genetic relationships between erythromycin-resistant strains were determined by multilocus variable tandem repeat analysis (MLVA). ResultsAll isolates were susceptible to penicillin, vancomycin, linezolid, and quinupristin–dalfopristin, but were resistant to tetracycline (96.6%, 141/146), erythromycin (28.1%, 41/146) and clindamycin (16.4%, 24/146). Among the 41 erythromycin-resistant GBS (ERGBS), the most common antimicrobial resistance gene was tetM detected in 92.7% (38/41) of the isolates followed by ermTR and ermB found in 65.8% (27/41) and 29.3% (12/41) of isolates, respectively. Of the 41 ERGBS, 95% (39/41) exhibited the constitutive MLSB phenotype, 2.4% (1/41) displayed inducible MLSB and 2.4% (1/41) had M phenotype. The erm methylase genes were widely related to MLSB phenotype isolates, while the mefA gene was associated with M phenotype. MLVA analysis performed on the 41 ERGBS revealed that 34 MLVA types (MTs). MLVA analysis showed that infections due to ERGBS have been caused by a variety of genotypes, suggesting that ERGBS were clonally unrelated and dissemination of these isolates was not due to a clonal outbreak. ConclusionCareful usage of macrolide antibiotics in therapy, continued surveillance of resistance rate and appropriate infection control measures can help to reduce spreading of resistance isolates.

Clostridioides difficile Ribotype 027 (RT027) Outbreak Investigation Due to the Emergence of Rifampicin Resistance Using Multilocus Variable-Number Tandem Repeat Analysis (MLVA).

ObjectiveThe aim of this study was Clostridioides difficile outbreak investigation due to the emergence of rifampicin resistant ribotype 027 (RT 027) fecal isolates from patients of Polish tertiary care hospital between X. 2017 and II. 2018 using multilocus variable tandem repeat analysis (MLVA).Materials and MethodsTwenty-nine C. difficile fecal isolates from patients of tertiary care hospital in Southern Poland were ribotyped and analyzed by MLVA. Multiplex PCR (mPCR) for genes encoding GDH (gluD), toxins A (tcdA)/ B (tcdB), 16S rDNA and binary toxin genes (ctdA and ctdB) was performed. The antibiotic susceptibility profile was determined by E-test.ResultsThe A, B and binary toxins encoding genes were detected in all 29 C. difficile strains which were sensitive to metronidazole, vancomycin and were resistant to erythromycin, clindamycin, and moxifloxacin; resistance to imipenem demonstrated 97%, to rifampicin – 45% isolates. C. difficile strains could be grouped by MLVA into 5 distinct clusters, and the largest cluster II contains 16 strains. The comparison of rifampicin GM MIC of cluster II (n=16 strains) with all others (n=13) showed that strains from clusters I, III, IV and V possessed significantly (p <0.005) higher GM MIC and were more resistant to rifampicin.ConclusionMLVA analysis proved transmission and recognized outbreak due to multidrug-resistant RT 027 C. difficile among patients of tertiary care hospital in Southern Poland. The reason for this is probably the widespread occurrence of spores in the hospital environment, which includes, among others, neglect of hygienic procedures and epidemic supervision. High resistance to imipenem (97%) and to rifampicin (45%) among C. difficile RT 027 Silesian isolates is threatening and requires further studies to elucidate this phenomenon.

Mycobacterium caprae in a dairy farm in Northeast Italy.

Veneto region, Northeast Italy, has been declared officially free from bovine tuberculosis since 2008, although the disease is sporadically detected in association with cattle trade. In September 2015, bovine tuberculosis was detected in a dairy cattle farm of the region, in a holding with 69 animals. The herd underwent single intradermal tuberculin testing as part of the regional surveillance plan, and 24 animals resulted positive. Mycobacterium caprae was evidenced in 22 samples, further genotyped by PCR-based assays, as Allgäu type. Epidemiological investigation reported that sixteen animals were introduced from an officially tuberculosis free Member State in previous years. Nevertheless, spoligotyping and multilocus variable tandem repeat analysis (MLVA) indicated that M. caprae was strictly related to the strain circulating in 2007-2009 in Trento province, although no at-risk contacts were described. M. caprae is a zoonotic pathogen and further analyses are warranted in order to control its spread and impact on public health and animal trade.

Occurrence and characterization of quinolone resistant Escherichia coli from Norwegian turkey meat and complete sequence of an IncX1 plasmid encoding qnrS1.

Plasmid-mediated quinolone resistance (PMQR) is frequent among Escherichia coli from various food products and animals in several countries. The objective of this study was to characterize quinolone resistant E. coli (QREC) from Norwegian turkey meat regarding resistance profiles, genetic mechanisms for quinolone resistance, genetic relatedness, and to investigate whether PMQR genes were present. In total, 78 QREC were isolated by a selective method from 156 samples throughout 2013. Isolates were subjected to susceptibility testing, characterization of resistance mechanisms, serotyping, phylotyping and multi-locus variable-tandem repeat analysis (MLVA). All 78 isolates were resistant to ciprofloxacin, while 77 were also resistant to nalidixic acid. The nalidixic acid sensitive isolate had a resistance profile indicating the presence of a PMQR gene. Both PCR and whole genome sequencing confirmed the presence of a 47 304 kb IncX1 plasmid containing the qnrS1 gene. The mechanism conferring quinolone resistance in the remaining isolates was mediated by mutations in the quinolone resistance-determining region of the chromosomal gyrA gene and for most of the isolates also in the parC gene. Molecular typing by MLVA showed a high degree of genetic diversity, although four clusters dominated. Two clusters contained strains belonging to phylogroup D/serogroup O176, the third contained isolates of phylogroup B1/serogroup O19, whereas the fourth contained isolates of phylogroup B1/non-typeable serogroup. Isolates within the latter cluster had MLVA profiles identical to QREC isolated from day-old imported turkey parent animals investigated in a preliminary study at the Norwegian Veterinary Institute. This finding suggests that QREC obtained from turkey may have been introduced via import of breeding animals to Norway. This is the first time the qnrS1 gene is described from E. coli isolated from Norwegian turkey meat. Compared to available qnrS1 carrying plasmids in Genbank, the current IncX1 plasmid showed high degree of similarity to other IncX1 plasmids containing qnrS1 isolated from both Shigella flexneri and E. coli found in different geographical areas and sources. To conclude, this study showed that mutations in gyrA and parC are the main mechanism conferring quinolone resistance in E. coli isolated from Norwegian turkey meat, and that PMQR has not been widely dispersed throughout the E. coli population in Norwegian turkey.

Antibiotic resistance, ESBL genes, integrons, phylogenetic groups and MLVA profiles of Escherichia coli pathotypes isolated from patients with diarrhea and farm animals in south-east of Iran.

The aims of this study were to investigate the prevalence, antibiotic resistance, presence of class 1 and 2 integrons, Extended Spectrum β-Lactamases (ESBL) genes, phylogenetic group and epidemiological relationships of EPEC, ETEC and EHEC pathotypes isolated from patients with diarrhea and farm animals in south east region of Iran. A total of 671 diarrheagenic E. coli (DEC) were collected from stool samples of 395 patients with diarrhea and 276 farm cattles and goats. Presence of EPEC, ETEC and EHEC were identified using multiplex-PCR employing primers targeted the shiga toxin (stx), intimin (eae), bundle forming pili (bfp), and enterotoxins (lt and st) genes. The highest proportion of the patients (64%) were children under age 1-15 year (p ≤ 0.05). Among the isolates, atypical EPEC was detected in 26 patients and 14 animal stool samples, while typical EPEC was found in 2 cattles. ETEC isolates were detected in stools of 13 patients and 4 EHEC was identified in 3 goats and one cattle. The isolates were checked for susceptibility to 14 antibiotics. 50% (n = 13) of EPEC and 61.5% (n =8) of ETEC showed multi-drug resistance (MDR) profiles and one EPEC was found to be extensive drug resistant (XDR). In contrast, EHEC isolates were susceptible to the majority of antimicrobial agents. The MDR isolates were positive for blaTEM and blaCTX-M ESBL genes and carried class 1 integrons. Further study on the biofilm formation indicated that, 3 out of 4 EHEC isolates showed strong biofilm, while other pathotypes had either moderate, weak or no biofilm activity. Majority of EPEC isolates were belonged to phylogenetic group B1, all except one ETEC were classified as phylogenetic group A and two EHEC were belonged to phylogroup D, respectively. A multilocus variable tandem repeat analysis (MLVA) exhibited 22 distinct patterns. In conclusion, MLVA data showed high clonal diversity. Presence of EHEC in animal origins pose public health concern in this region.

Clostridioides difficile in the environment, food, animals and humans in southern Italy: Occurrence and genetic relatedness.

One hundred and thirty-eight C. difficile isolates from different sources (66 from the environment, 36 from animals, 9 from food and 27 from humans) were ribotyped by capillary electrophoresis PCR ribotyping (CE-PCR). A multilocus variable tandem repeat analysis (MLVA) was carried out on a sample subset. The most frequently isolated PCR ribotypes were 126 (15.9%), 078 (14.5%), 011/018 (11.6%), 014/020/077 (10.1%), and 010 (2.8%). In particular, strains of PCR ribotype 011/018 were isolated from human, raw milk and environmental samples. The hypervirulent PCR ribotype 027 was isolated from two human samples. The majority of the strains were toxigenic (34.1% showed the toxigenic profile A+B+CDT+ and 38.9% the profile A+B+CDT-). MLVA allowed to identify 4 clonal complexes of genetically related isolates: complex n. 1 grouped together human, environmental and food strains, whereas complex n. 3 included human and environmental isolates. The use of MLVA gave further evidence to the possible role of environment, animals and food as routes of transmission of C. difficile infections to human.

Pertussis Outbreak in a Primary School in China: Infection and Transmission of the Macrolide-resistant Bordetella pertussis.

A pertussis outbreak was studied in a primary school in Xi'an, China, in March 2016. The school consisted of 536 pupils 6-12 years of age who were divided into 12 classes of 6 grades (2 classes for each grade). The identified index case was an 11-year-old girl at class 2 of grade 5. Interview was conducted and nasopharyngeal swabs were taken from all pupils (N = 94) in the 2 classes of grade 5. Nasopharyngeal swabs were tested by both culture and polymerase chain reaction (PCR). Four culture- and 17 PCR-positive cases were identified in 94 pupils. Infection rate was significantly higher in class 2 compared with that in class 1 [37.0% (17/46) vs. 14.6% (7/48), χ(2) = 4.26, P < 0.05]. All Bordetella pertussis isolates were macrolide-resistant, harbored prn1/ptxP1/fim3-1 as previously reported and belonged to multilocus variable tandem repeat analysis type MLVA 195. Of the 17 DNAs positive for diagnostic PCR, 12 were also positive for 23S ribosomal RNA PCR. All the 12 DNAs had the A2047G mutation of 23S rRNA gene of B. pertussis. This study described a pertussis outbreak caused by macrolide-resistant B. pertussis in a primary school and indicated that close contact of index case causes the bacterial transmission.

Dynamics of cholera epidemics from Benin to Mauritania.

BackgroundThe countries of West Africa are largely portrayed as cholera endemic, although the dynamics of outbreaks in this region of Africa remain largely unclear.Methodology/Principal findingsTo understand the dynamics of cholera in a major portion of West Africa, we analyzed cholera epidemics from 2009 to 2015 from Benin to Mauritania. We conducted a series of field visits as well as multilocus variable tandem repeat analysis and whole-genome sequencing analysis of V. cholerae isolates throughout the study region. During this period, Ghana accounted for 52% of the reported cases in the entire study region (coastal countries from Benin to Mauritania). From 2009 to 2015, we found that one major wave of cholera outbreaks spread from Accra in 2011 northwestward to Sierra Leone and Guinea in 2012. Molecular epidemiology analysis confirmed that the 2011 Ghanaian isolates were related to those that seeded the 2012 epidemics in Guinea and Sierra Leone. Interestingly, we found that many countries deemed “cholera endemic” actually suffered very few outbreaks, with multi-year lulls.Conclusions/SignificanceThis study provides the first cohesive vision of the dynamics of cholera epidemics in a major portion of West Africa. This epidemiological overview shows that from 2009 to 2015, at least 54% of reported cases concerned populations living in the three urban areas of Accra, Freetown, and Conakry. These findings may serve as a guide to better target cholera prevention and control efforts in the identified cholera hotspots in West Africa.

Five-year surveillance of human tuberculosis caused by Mycobacterium bovis in Bologna, Italy: an underestimated problem.

Human tuberculosis (TB) caused by Mycobacterium bovis surveillance is affected by a lack of data. The aims of the present study were: (i) to estimate the proportion of human TB caused by M. bovis over a period of 5 years in Bologna, Northern Italy, which, like most Western European countries, has been declared bovine TB-free; (ii) to compare the genetic profiles of M. bovis strains identified in humans with those circulating in cattle in the last 15 years in Italy. Among 511 TB patients, the proportion of human TB caused by M. bovis was 1·76%, significantly associated to extra-pulmonary localization (P = 0·004) and to being elderly (P < 0·001) and Italy-born (P = 0·036). The molecular epidemiology analysis by spoligotyping and Multilocus Variable Tandem Repeat Analysis confirmed that most M. bovis strains from Italy-born patients matched those circulating in cattle herds in Italy between 2001 and 2016. Two cases of Mycobacterium bovis BCG infection were also characterized. In conclusion, the rate of human TB caused by M. bovis was not negligible, highlighting the relevance of molecular typing in evaluating the effectiveness of programmes designed to eradicate TB in cattle in Italy.

The recognition and characterisation of Finnish Clostridium difficile isolates resembling PCR-ribotype 027.

To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods. Twenty-eight C. difficile RT027-like isolates and 11 RT027 comparative strains were characterised by capillary-electrophoresis (CE) ribotyping, multi-locus variable tandem-repeats analysis (MLVA), multi-locus sequence typing (MLST), and sequencing of tcdC and gyrA gene fragments. Susceptibility to moxifloxacin was determined by E-test. Of 28 RT027-like isolates, seven RTs (016, 034, 075, 080, 153, 176 and 328), three WEBRIBO types (411, 475, AI-78) and three new profiles (F1-F3) were identified. MLVA revealed six clonal complexes (RTs 016, 027, 176 and F3). MLST showed eleven sequence types (1, 41, 47, 67, 95, 191,192, 223, 229, 264 and new ST). Twenty-two isolates (RTs 016, 080, 176, 328, F1, F2, F3 and WRTAI-78) carried Δ117 in the tcdC gene. Isolates of RTs 016, 027 and 176 were moxifloxacin resistant and harboured Thr82Ile in the GyrA. Our results show a high diversity within 28 Finnish RT027-like C. difficile isolates, with twelve CE-ribotyping profiles and eleven STs. MLVA revealed the regional spread of RTs 016, 027, 176 and F3. The presence of Δ117 in the tcdC gene in eight non-027 RTs highlights the importance of careful interpretation of the results from molecular systems targeting this site in the genome of C. difficile and the need of strain typing for epidemiological purposes.

Two Clusters of Fluoroquinolone and Clindamycin-Resistant Clostridium difficile PCR Ribotype 001 Strain Recognized by Capillary Electrophoresis Ribotyping and Multilocus Variable Tandem Repeat Analysis.

To perform a retrospective analysis of the high occurrence of Clostridium difficile infection in the surgical department of a Czech tertiary care hospital and to identify weaknesses in C. difficile infection (CDI) prevention and control policies. Clinical and epidemiological data on eleven CDI cases were collected. C. difficile isolates were characterized by capillary electrophoresis ribotyping, multilocus variable tandem repeat analysis (MLVA), gyrA gene fragment sequencing, and erm(B) fragment PCR amplification. Antibiotic susceptibility to metronidazole, vancomycin, ciprofloxacin, moxifloxacin, and clindamycin was tested. Eleven CDI cases were caused by C. difficile PCR ribotype 001 strains. These strains revealed two different MLVA profiles with 11 tandem repeat differences. All isolates were susceptible to metronidazole and vancomycin and resistant to ciprofloxacin (MIC ≥32 mg/L), moxifloxacin (MIC ≥32 mg/L), and clindamycin (MIC ≥256 mg/L). All isolates revealed amino acid substitution Thr82Ile, in the GyrA and were erm(B) negative. Two fluoroquinolone and clindamycin-resistant C. difficile PCR ribotype 001 strain clusters occurred at one of the surgical departments of a tertiary care hospital. Ineffective decontamination with suboptimal concentration and time of exposure of sporicidal disinfectants may have resulted in C. difficile transmission.

Molecular Epidemiology and Antibiotic Susceptibility of Vibrio cholerae Associated with a Large Cholera Outbreak in Ghana in 2014

BackgroundGhana is affected by regular cholera epidemics and an annual average of 3,066 cases since 2000. In 2014, Ghana experienced one of its largest cholera outbreaks within a decade with more than 20,000 notified infections. In order to attribute this rise in cases to a newly emerging strain or to multiple simultaneous outbreaks involving multi-clonal strains, outbreak isolates were characterized, subtyped and compared to previous epidemics in 2011 and 2012.Methodology/Principal FindingsSerotypes, biotypes, antibiotic susceptibilities were determined for 92 Vibrio cholerae isolates collected in 2011, 2012 and 2014 from Southern Ghana. For a subgroup of 45 isolates pulsed-field gel electrophoresis, multilocus sequence typing and multilocus-variable tandem repeat analysis (MLVA) were performed. Eighty-nine isolates (97%) were identified as ctxB (classical type) positive V. cholerae O1 biotype El Tor and three (3%) isolates were cholera toxin negative non-O1/non-O139 V. cholerae. Among the selected isolates only sulfamethoxazole/trimethoprim resistance was detectable in 2011, while 95% of all 2014 isolates showed resistance towards sulfamethoxazole/trimethoprim, ampicillin and reduced susceptibility to ciprofloxacin. MLVA achieved the highest subtype discrimination, revealing 22 genotypes with one major outbreak cluster in each of the three outbreak years. Apart from those clusters genetically distant genotypes circulate during each annual epidemic.Conclusions/SignificanceThis analysis suggests different endemic reservoirs of V. cholerae in Ghana with distinct annual outbreak clusters accompanied by the occurrence of genetically distant genotypes. Preventive measures for cholera transmission should focus on aquatic reservoirs. Rapidly emerging multidrug resistance must be monitored closely.

Molecular characterization of the circulating Bacillus anthracis in Jordan.

To understand the biomolecular charcteristics of Bacillus anthracis in Jordan, 20 blood smear slides from dead animals with suspected anthrax were analyzed using conventional and molecular approaches. All slides were positive for B. anthracis by conventional staining but no growth of the organism on selective media was detected. However, of the 20 samples, 16 were B. anthracis DNA-positive using polymerase chain reaction (PCR). Seven samples provided enough quantity and quality of DNA, and their multilocus variable tandem repeat analysis (MLVA)-15 loci analysis revealed two different genotypes. All genotypes were belonging to A.B..r. 008/009 which is very common in Asia and Europe. Single nucleotide repeat (SNR) analysis revealed that there were no sub genotypes. Molecular diagnosis of animal anthrax in Jordan is not used routinely; henceforth, official diagnosis of anthrax is based on the observation of the slides by optical microscope and this can often cause reading errors. Therefore, the prevalence of the disease in Jordan might be slightly lower than that reported by the official bodies.

Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia

A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004–2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes – emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies.

Genotyping techniques in Yersinia pestis

There are different genotyping techniques, including partial genomic information-based methods such as pulsed-filed gel electrophoresis (PFGE), multilocus variable tandem repeat analysis (MLVA), different region (DFR) analysis etc., and whole genome-based comparative genomics, available for bacterial analysis. Many of them have been successfully applied in Yersinia pestis. In accompany with one paper on Yersinia pestis plasmid profiles and one review on genotyping analysis in Yersinia pestis, published in the same issue, this review briefed the different genotyping techniques in focus of their applications. Key words: Yersinia pestis; Genotype; Genotyping techniques

E. coli Bacteremia Strains - High diversity and Associations with Agerelated Clinical Phenomena

Advanced age is associated with an increased proportion of Escherichia coli in bacteremia as well as an increased risk of death from E. coli blood stream infection. Age-associated differences in normal flora E. coli indicate that elderly persons may be diseased by other groups of E. coli than do younger patients. We studied a historical cohort of 212 patients with community-acquired E. coli bacteremia. The bacterial strains were tested for antimicrobial resistance and analyzed by a generic Multi-Locus Variable-Tandem Repeats Analysis (MLVA). The available 212 strains showed a great diversity, and clustered into ten different MLVA-type complexes (MTC). MTC-b, containing 97 of the strains, was associated with ≥1 comorbid illness (OR 2.02, 95% CI 1.12-3.64), and with to ≥1 atypical symptom (OR 0.46, 95% CI 0.27-0.80). MTC-c, containing 31 strains, was associated with urinary origin of infection (OR 3.28, 95% CI 1.345-8.00) and was preventive against gastrointestinal origin of infection (OR 0.11, 95% CI 0.01-0.83). MTC-g, containing only eight strains, was associated with leukopenia (OR 6.43, 95% CI 1.15-36.00). The strains showed low level of antimicrobial resistance. Fifteen of the 212 patients (7.1%) died within 14 days after admission to hospital. Neither MTC nor antimicrobial resistance was associated with hospital mortality. In conclusion, our study showed a great degree of diversity of the strains and that one of the MTCs was associated with age-related clinical phenomena. None of the MTCs were associated with outcome, indicating that patient characteristics are more important than microbial characteristics.

Progress toward Rapid and Accurate Staphylococcus aureus Strain Typing

Staphylococcus aureus , especially methicillin-resistant S. aureus (MRSA),1 continues to be one of the most important bacterial pathogens in humans and is responsible for a constantly increasing number of nosocomial and community-acquired infections. Bacterial strain typing, or subspecies identification, has become an important clinical tool for investigating suspected outbreaks and evaluating nosocomial transmission. Adequate and precise typing of S. aureus isolates that permits the monitoring of local outbreaks and wider-scale dissemination of specific dangerous clones is of great concern (1)(2)(3). Similar to typing in other microbial organisms, S. aureus typing has been greatly influenced and improved by molecular techniques. Differences in the various molecular methods used to define different genomic characteristics greatly affect their essential parameters, including discriminatory power and test turnaround time (1)(2)(3). The most important methods currently used for MRSA typing are pulsed-field gel electrophoresis (PFGE) (4)(5)(6)(7), multilocus sequence typing (MLST) (8)(9), spa (staphylococcal surface protein A) typing (1)(6)(10)(11), multilocus variable tandem repeat analysis (MLVA) (12)(13), and repeat-element PCR (rep-PCR) typing (7)(14)(15). These methods have been used to investigate the evolution of the MRSA clones that have emerged since the 1960s and to study their worldwide dissemination. Tremendous progress in molecular-typing methods has been made, with optimization and standardization of sequence-based technologies offering broad applicability and high throughput; however, no single S. aureus typing method has yet provided rapid and reliable information that is sufficient to permit prompt action to control infections. Because S. aureus has a highly clonal population structure, many different chromosomal markers indexing polymorphisms …