Emergence of methicillin-resistant,vancomycin-intermediate Staphylococcus aureus among patients associated with group A Streptococcal pharyngitis infection in southern India

Beyond Staphylococcus aureus being an etiological agent for several serious clinical complications, the foot prints of S. aureus in pharyngitis infection has also been recently recognized. With due response to the fact, a prospective study was conducted between 2009 and 2010 to describe the molecular epidemiology of S. aureus in throat swabs of pharyngitis patients. A total of 63 methicillin-resistant S. aureus (MRSA) and 102 methicillin-susceptible S. aureus (MSSA) isolates were recovered from 265 throat swabs, representing a community-acquired outpatient population from Tamil Nadu, India. Molecular characterization of MRSA was done by two conventional multiplex PCR assays including Panton-Valentine leukocidin (PVL), mecA and nuc genes, and staphylococcal cassette chromosome mec (SCCmec) typing. Among 165 S. aureus isolates, methicillin resistance was observed in 38.2% (n = 63), in which 69.8% (n = 44/63) of the MRSA along with 55.9% (n = 57/102) of MSSA harbored PVL toxin genes. SCCmec typing showed 50.8% of isolates as SCCmec V (n = 32), 44.4% as SCCmec III (n = 28), and 1.6% as SCCmec types I, II and IVa (n = 1). Multilocus sequence typing performed for 26 selected MRSA isolates resulted in 12 different sequence types (ST), including a novel ST2129/SCCmec III, PVL-positive. Ten MRSA isolates were categorized as ST772 (38.5%)/SCCmec V, PVL-positive, and three isolates as ST368 (11.5%)/SCCmec III, PVL-negative. Though the prominent clones of ST772/SCCmec V were multidrug-susceptible worldwide, they were highly multidrug-resistant in the current study, including four clones intermediate to vancomycin. Totally, 10 (15.9%) out of 63 MRSA isolates were documented as vancomycin-intermediate S. aureus (VISA). Collectively, the present study for the first time portrayed the high prevalence of active MRSA pharyngitis infection and also emphasizes an alarming need for discrimination of pharyngeal-asymptomatic carriers of S. aureus from those with an active S. aureus pharyngitis infection.     

Molecular epidemiology and antibiotic resistance of methicillin-resistant Staphylococcus aureus circulating in the Russian Federation

The aim of this study was to investigate the patterns of antimicrobial resistance and molecular features of methicillin-resistant Staphylococcus aureus (MRSA) isolates in Russia. Isolates recovered from hospital patients (n = 480), healthy medical personnel (n = 25), and healthy carriers (n = 13) were included in the study. Hospital-acquired MRSA (HA-MRSA) demonstrated high resistance to ciprofloxacin, gentamicin, and chloramphenicol (76%–92%), moderate – to tetracycline, erythromycin, clindamycin, and rifampicin (38%–54%), and low – to fusidic acid, co-trimoxazole, mupirocin, and daptomycin (2%–7%). Elevated MIC (2.0 μg/ml) of vancomycin was detected in 26% of isolates. All isolates were susceptible to linezolid and tigecycline. Multilocus sequence typing (MLST) revealed that CC8 isolates (ST8 + ST239) constituted 83.1% of HA-MRSA and that this genetic lineage dominated in all regions from Krasnoyarsk to Saint Petersburg. A local ST239 variant harboring the tst gene (ST239_Kras) was detected in Krasnoyarsk. The other HA-MRSA isolates belonged to clonal complex 5 (CC5) (21 isolates, 12.2%) and CC22 (2, 1.2%). The majority of CC5 isolates were affiliated with sequence type 228 (ST228) and were characterized with decreased susceptibility to ceftaroline (MIC = 2 μg/ml). We also detected, for the first time in Russia, livestock-associated MRSA (LA-MRSA) from clusters CC398 and CC97 in humans. Among the 2053 healthy persons screened for nasal carriage of S. aureus, the bacteria were isolated from 426 (21%); among them, 13 carried isolates identified as community-associated MRSA (CA-MRSA). Eleven of 13 CA-MRSA isolates belonged to ST22 (spa types t223, t3243, and t3689; SCCmec types IVa and IVc, agr type I, tst-positive) and were similar to the EMRSA-15/Middle Eastern variant (Gaza strain).     

Molecular analysis and the toxin,MSCRAMM, and biofilm genes of methicillin-resistant Staphylococcus aureus strains isolated from pemphigus wounds: A study based on SCCmec and dru typing

IntroductionPemphigus is a chronic autoimmune blistering disease. Pemphigus blisters can damage the natural skin barrier and increase the risk of life-threatening conditions. Colonization of pemphigus wounds with methicillin-resistant Staphylococcus aureus (MRSA) prolongs wound healing and increases mortality rate. Assessing MRSA prevalence, types, and toxin and adhesion genes can facilitate the detection of MRSA strains which cause infections, selection of appropriate treatments, and healing of pemphigus wounds. This study aimed to determine the SCCmec, the direct repeat unit (dru) types (dts), and the toxin, MSCRAMM, and biofilm genes of MRSA strains isolated from pemphigus wounds.MethodsIn this cross-sectional study, 118 S. aureus isolates were gathered from 118 patients with pemphigus. MRSA detection was performed using the mecA gene. Using the polymerase chain reaction method, all MRSA isolates were assessed for the presence of the sea, seb, sec, tst, eta, pvl, hla, hlb, MSCRAMM, and ica genes. Typing and subtyping were performed through respectively SCCmec typing and dru typing methods. The Bionumerics software was used for analyzing the data and drawing the minimum spanning tree.FindingsFrom 118 S. aureus isolates, 51 were MRSA. SCCmec typing revealed the prevalence of SCCmec II with a prevalence of 64.7% (33 out of 51 isolates) and SCCmec III with a prevalence of 35.3% (18 out of 51 isolates). Dru typing indicated seven dts, namely dts 10a, 10g, 10m, 13i, 8h, 8i, and 9ca in two main clusters. The dt9ca was a new dru type and was registered in the dru-typing database (www.dru-typing.org). The prevalence rates of the hla, sea, and sec genes in MRSA isolates were respectively 54.9%, 27.4%, and 1.9%, while the hlb, seb, eta, and pvl genes were not detected at all. Only one MRSA with SCCmec III and dt10a carried the tst encoding gene. MSCRAMM gene analysis revealed the high prevalence of the eno (31.3%) and the fib (21.5%) genes. The prevalence rates of the icaA and icaD biofilm formation genes were 3.9% and 5.8%, respectively. There were no significant differences between the two detected SCCmec types and between the two detected dts clusters respecting the prevalence of the encoding genes of virulence factors and MSCRAMMs.ConclusionThe toxin genes hla and sea are prevalent among MRSA strains with SCCmec II and III isolated from pemphigus wounds. The most prevalent dts are dt10a and dt10g among MRSA with SCCmec III and dt8h and dt8i among MRSA with SCCmec II.     

Prevalence and genetic diversity of arginine catabolic mobile element (ACME) in clinical isolates of coagulase-negative staphylococci: Identification of ACME type I variants in Staphylococcus epidermidis

Arginine catabolic mobile element (ACME), a genomic island consisting of the arc and/or opp3 gene clusters found in staphylococcal species, is related to increased bacterial adaptability to hosts. Staphylococcus epidermidis is considered a major ACME reservoir; however, prevalence and genetic diversity of ACME in coagulase-negative staphylococci (CNS) have not yet been well characterized for clinical isolates in Japan. A total of 271 clinical isolates of CNS in a Japanese hospital were investigated for the presence and genotype of ACME and SCCmec. The prevalence of ACME-arcA was significantly higher (p < 0.001) in S. epidermidis (45.8%) than in other CNS species (3.7%). ACME in S. epidermidis isolates (n = 87) were differentiated into type I (n = 33), variant forms of type I (ΔI, n = 26) newly identified in this study, type II (n = 6), and type ΔII (n = 19). ACME-type ΔI, which were further classified into three subtypes, lacked some genetic components between the arc and opp3 clusters in archetypal type I, whereas the arc and opp3 clusters were intact. The arc cluster exhibited high sequence identity (95.8–100%) to that of type I ACME; in contrast, the opp3 cluster was highly diverse, and showed relatively lower identities (94.8–98.7%) to the identical regions in type I ACME. Twenty-one isolates of ΔI ACME-carrying S. epidermidis possessed SCCmec IVa and belonged to ST5 (clonal complex 2). Phylogenetic analysis revealed that isolates harboring ACME ΔI in this study clustered with previously reported S. epidermidis strains with other lineges, suggesting that S. epidermidis originally had some genetic variations in the opp3 cluster. In summary, ACME type ΔI, a truncated variant of ACME-I, was first identified in S. epidermidis, and revealed to be prevalent in ST5 MRSE clinical isolates with SCCmec IVa.     

Genetic basis of antimicrobial resistance and clonal dynamics of carbapenem-resistant Acinetobacter baumannii sequence type 191 in a Korean hospital

This study investigated the genetic basis of antimicrobial resistance and the epidemiological characteristics of 125 carbapenem-resistant Acinetobacter baumannii (CRAB) isolates collected from 2011 to 2012 in a Korean hospital. All CRAB isolates showed an extensively drug-resistant phenotype, but were susceptible to tigecycline. The bla_OXA − 23 and armA genes were mainly responsible for resistance to carbapenems and aminoglycosides, respectively. Four colistin-resistant CRAB isolates with different pulsotypes were identified. All four colistin-resistant isolates had a deletion at nucleotide 776 in lpxA, while one also had an insertion at nucleotide 732 in lpxA. All CRAB isolates belonged to three sequence types (STs): ST191 (n = 118), ST208 (n = 6), and ST436 (n = 1), but were classified into 33 arbitrary pulsotypes. Of the CRAB ST191 isolates, two main arbitrary pulsotypes 5 (n = 20) and 18 (n = 17) emerged sequentially, but were not clonally related to CRAB isolates collected from 2009 to 2010 in the same hospital. Furthermore, of the two main pulsotypes identified among CRAB ST191 isolates from 2009 to 2010, one was clonally related to sporadic CRAB ST191 isolates from 2011 to 2012, but the other was not related to any CRAB isolate from 2011 to 2012. In conclusion, this study shows the clonal dynamics of CRAB ST191 isolates in a Korean hospital during the last four years.     

MRSA Pediatric clone expressing ermC plus lnuA genes causing nosocomial transmission and healthcare workers colonization in a neonatal intensive care unit

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of both nosocomial and community-acquired infections. We describe an outbreak caused by the MRSA Pediatric clone expressing an unusual lincosamide resistant phenotype. Between January and May 2006, an MRSA outbreak was detected at the Neonatal Unit of Hospital Interzonal General de Agudos “Evita”, Buenos Aires Province, Argentina that affected ten patients. Seven isolates from seven patients plus five MRSA recovered from health care workers (nasal carriage) were studied. Two phenotypes were observed: (i) ELCi (10), resistance to erythromycin and lincomycin and inducible resistance to clindamycin; (ii) ELiCi (2), resistance to erythromycin and inducible resistance to lincomycin and clindamycin. All 12 MRSA were resistant to oxacillin, erythromycin and gentamicin. Isolates expressing the ELCi-phenotype showed lincomycin MIC values between 16 and 32 mg/L, while the remaining 2 isolates with ELiCi-phenotype presented a MIC value of 0.5 mg/L. No differences were observed between the clindamycin MIC values in both phenotypes, ranging 0.25–0.5 mg/L. Isolates showing ELCi-phenotype harbored ermC plus lnuA genes, and the other two only ermC gene. All 12 isolates were genetically related and belonged to the Pediatric clone (ST100) harboring a new variant of SCCmecIV. This is the first MRSA outbreak expressing an unusual ELCi phenotype due to a combination of ermC plus lnuA genes.     

Performance of a PCR assay for the rapid identification of the Klebsiella pneumoniae ST258 epidemic clone in Latin American clinical isolates

The worldwide dissemination of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae ST258 demands a rapid PCR-based typing method to detect unique genes of the ST258 clone. This study evaluates a PCR developed by Adler et al. (2014) for the detection of ST258 in K. pneumoniae clinical isolates centered on the identification of the pilv-I and prp genes. We tested 143 clinical isolates from Argentina (n = 109), Chile (n = 1), Colombia (n = 1), Costa Rica (n = 2), Ecuador (n = 5), El Salvador (n = 2), Nicaragua (n = 5), Panamá (n = 2), Paraguay (n = 2), Perú (n = 3) and Trinidad and Tobago (n = 11) recovered from 2006 to 2015. bla_KPC, pilv-l and prp genes were detected by PCR and sequenced by standard procedures. ST258 and non-ST258 were defined by PFGE and/or MLST. Isolates were grouped according to PFGE patterns: 58 were compatible with ST258 (group 1) and 85 with non-ST258 (group 2). MLST study was done on an arbitrary selection of isolates. The pilv-l gene was present only in ST258 isolates, regardless of the presence of the bla_KPC gene. Results for the prp gene were variable. Its presence did not define ST258. The pilv-I PCR had a sensitivity and specificity of 100%, respectively, for the detection of ST258 in the isolates under investigation. Given our findings, the pilv-I PCR could replace more time and resource consuming methods, allowing for more rapid detection of the circulating high risk K. pneumoniae clone ST258 in Latin American (LA) countries.     

Genotyping of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) in a tertiary care centre in Mysore,South India: ST2371-SCCmec IV emerges as the major clone

The burden of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) is on the rise in population and clinical settings on account of the adaptability and virulence traits of this pathogen. We characterized 45 non-duplicate CA-MRSA strains implicated mainly in skin and soft tissue infections (SSTIs) in a tertiary care hospital in Mysore, South India. All the isolates were genotyped by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing, accessory gene regulator (agr) typing, and multi-locus sequence typing (MLST). Four sequence types (STs) belonging to three major clonal complexes (CCs) were identified among the isolates: CC22 (ST2371 and ST22), CC1 (ST772) and CC8 (ST8). The majority (53.3%) of the isolates was of the genotype ST2371-t852-SCCmec IV [sequence type-spa type-SCCmec type], followed by ST22-t852-SCCmec IV (22.2%), ST772-t657-SCCmec V (13.3%) and ST8-t008-SCCmec IV (11.1%). ST237I, a single locus variant of ST22 (EMRSA-15 clone), has not been reported previously from any of the Asian countries. Our study also documents for the first time, the appearance of ST8-SCCmec IV (USA300) strains in India. Representative strains of the STs were further analyzed by pulsed field gel electrophoresis (PFGE). agr typing detected type I or II alleles in the majority of the isolates. All the isolates were positive for the leukotoxin gene, pvl (Panton–Valentine leukocidin) and the staphylococcal enterotoxin gene cluster, egc. Interestingly, multidrug resistance (resistance to ⩾3 classes of non-beta-lactam antibiotics) was observed in 77.8% (n = 35) of the isolates. The highest (75.5%) resistance was recorded for ciprofloxacin, followed by erythromycin (53.3%), and quinupristin–dalfopristin (51.1%). Inducible clindamycin-resistance was identified in 37.7% of the isolates and it was attributed to the presence of erm(A), erm(C) and a combination of erm(A) and erm(C) genes. Isolates which showed a phenotypic pattern of M^R/L^S (macrolide-resistance/lincosamide-sensitivity) harbored the msr(A) gene. In conclusion, we report a high rate of multidrug resistance among Indian strains of CA-MRSA and the emergence of the lineages ST2371 and ST8 in India.     

Prevalence of CTX-M extended-spectrum beta-lactamases and sequence type 131 in Korean blood,urine, and rectal Escherichia coli isolates

A high proportion of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli are of the ST131 lineage, but there are few estimates of ST131 prevalence among ESBL-negative E. coli. Without this information, it is difficult to evaluate the contribution of the ST131 lineage to the emergence and spread of ESBL E. coli. A total of 1658 E. coli isolates were collected at Gachon University Gil Medical Center in Korea from 2006 to 2008. The antibiotic resistance profile was determined for all isolates, and ESBL-positive isolates were screened for the presence of CTX-M-type ESBLs. All ESBL-positive (n = 84) and a representative sample of ESBL-negative (n = 100) isolates were screened for O25b-ST131 using a PCR-based assay. The isolates were further classified on the basis of fumC and fimH types, which allowed for a comparison of the two typing methods. 5.7% of isolates were ESBL-positive, 87% of which contained CTX-M-type ESBLs. There was no significant difference in the prevalence of ST131 between ESBL-positive and -negative groups; 14% of ESBL-positive isolates and 9% of tested ESBL-negative isolates were ST131 by CH-typing. ST131-positive isolates harbored CTX-M-1-group ESBLs (including CTX-M-15) more frequently than other CTX-M types, and exhibited greater levels of antibiotic resistance than non-ST131 isolates. Furthermore, a number of isolates identified as O25b-ST131 by PCR corresponded to non-ST131 sequence types by CH-typing, emphasizing the need to consider the testing method when comparing reported prevalences of ST131.     

Population structure of Legionella spp. from environmental samples in Gabon, 2013

Aquatic environments are the most important source for Legionella spp. infections such as Legionnaires’ disease and Pontiac fever. The reservoirs of Legionella spp. are mostly unclear in sub-Saharan Africa. The aim of this study, conducted in 2013, was to identify geographical areas of an increased risk for exposure to Legionella spp., and to describe the population structure of Legionella spp. from different water sources in a cross-sectional study in Gabon. Fresh water samples (n = 200) were cultured on Legionella selective agar; species were confirmed by MALDI-TOF, a Legionella pneumophila specific real-time PCR and 16S RNA gene sequencing. Serogroups were identified by agglutination test. The population structure was assessed by multilocus sequence typing (MLST).Legionella spp. isolates (n = 29) were frequently found in the hospital setting particularly in hot water systems. Open water bodies (i.e. rivers, lakes) were not contaminated with Legionella spp. Isolated L. pneumophila mainly belonged to serogroups 2–14 (n = 19) and MLST sequence type ST1, ST75 (and related STs) and ST1911.In conclusion, hospitalized patients might have an increased risk to become infected with Legionella spp. in the studied areas in Gabon, particularly if they have risk factors such as comorbidities. Both broadly extended (ST1, ST75) and local lineages (ST1911) were present in our setting.     

A combined multi-virulence-locus sequence typing and Staphylococcal Cassette Chromosome mec typing scheme possesses enhanced discriminatory power for genotyping MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major threat to human populations worldwide. Knowing the extent of MRSA genetic diversity within a healthcare facility may provide important insights into the epidemiology of this important pathogen. MRSA isolates recovered from nasal swabs of patients entering the Intensive Care Unit of the Penn State Milton S. Hershey Medical Center, USA, from 2008 to 2009 were genotyped using Staphylococcal Cassette Chromosome mec (SCCmec), multilocus sequence typing (MLST) and a newly developed multi-virulence-locus sequence typing (MVLST) scheme. Sequence data for seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi and yqiL) and six virulence genes (alt, essC, geh, hlgA, htrA and sdrC) were used for MLST and MVLST analyses, respectively. MLST identified 12 sequence types (STs) within the hospital isolates. One ST designated ST5 was the most common subtype (38.8%) followed by ST105 (22.4%) and ST8 (16.4%). In contrast, MVLST identified 29 STs (Virulence Types, VTs) from the same set of isolates, with VT6 (32.8%) being the predominant subtype followed by VT9 (8.9%) and VT2 (8.9%). Subsequent analysis of 25 MRSA isolates associated with an outbreak at a Pennsylvania state prison revealed all isolates were VT2 and SCCmec type IVa. These results suggest that a combination of MVLST and SCCmec typing may clarify the epidemiology of MRSA. Additional research with a more diverse set of strains and correlation with conventional epidemiologic data are needed to validate this new subtyping strategy.     

The predominance of Ethiopian specific Mycobacterium tuberculosis families and minimal contribution of Mycobacterium bovis in tuberculous lymphadenitis patients in Southwest Ethiopia

BackgroundEthiopia has an extremely high rate of extrapulmonary tuberculosis, dominated by tuberculous lymphadenitis (TBLN). However, little is known about Mycobacterium tuberculosis complex (MTBc) lineages responsible for TBLN in Southwest Ethiopia.MethodsA total of 304 MTBc isolates from TBLN patients in Southwest Ethiopia were genotyped primarily by spoligotyping. Isolates of selected spoligotypes were further analyzed by 15-loci mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) (n = 167) and qPCR-based single nucleotide polymorphism (n = 38). Isolates were classified into main phylogenetic lineages and families by using the reference strain collections and identification tools available at MIRU-VNTRplus data base. Resistance to rifampicin was determined by Xpert MTB/RIF.ResultsThe majority of isolates (248; 81.6%) belonged to the Euro-American lineage (Lineage 4), with the ill-defined T and Haarlem as largest families comprising 116 (38.2%) and 43 (14.1%) isolates respectively. Of the T family, 108 isolates were classified as being part of the newly described Ethiopian families, namely Ethiopia_2 (n = 44), Ethiopia_3 (n = 34) and Ethiopia_H₃₇Rv-like (n = 30). Other sub-lineages included URAL (n = 18), S (n = 17), Uganda I (n = 16), LAM (n = 13), X (n = 5), TUR (n = 5), Uganda II (n = 4) and unknown (n = 19). Lineage 3 (Delhi/CAS) was the second most common lineage comprising 44 (14.5%) isolates. Interestingly, six isolates (2%) were belonged to Lineage 7, unique to Ethiopia. Lineage 1 (East-African Indian) and Lineage 2 (Beijing) were represented by 3 and 1 isolates respectively. M. bovis was identified in only two (0.7%) TBLN cases. The cluster rate was highest for Ethiopia_3 isolates showing clonal similarity with isolates from North Ethiopia. Lineage 3 was significantly associated with rifampicin resistance.ConclusionsIn TBLN in Southwest Ethiopia, the recently described Ethiopia specific Lineage 4 families were predominant, followed by Lineage 3 and Lineage 4-Haarlem. The contribution of M. bovis in TBLN infection is minimal.     

Evaluation of a new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clonal complexes circulating in Brazil

Dissemination of carbapenem-resistant Acinetobacter baumannii (CRAB) is mainly due to the spread of clonal lineages, particularly those included into the clonal complexes (CC) CC1, CC2, CC15, CC25, and CC79. We evaluated the usefulness of a recently modified PCR-based trilocus sequence-based typing (m3LST) in comparison with the standard multilocus sequence typing (MSLT) of 7 housekeeping genes as per the Institute Pasteur Scheme to assign the clonal complexes in CRAB. A collection of 78 CRAB isolated from 67 different Brazilian health institutions was submitted to both methodologies, and concordance rate was calculated. The collection studied included mainly isolates belonging to endemic Brazilian Clonal Complexes (CC1, CC15, CC25 and CC79, n = 72, 92.3%) but also singletons sequence types (ST) with low prevalence in the country (ST107, ST113, ST188, ST317, ST584, ST733, n = 6; 7.7%). The m3LST correctly assigned all the isolates into the main CC responsible for the CRAB dissemination in Brazil. All the singletons ST were not misidentified as prevalent lineages. The PCR-based m3LST is a powerful tool to investigate molecular epidemiology of A. baumannii representative of prevalent Brazilian clonal complexes 1, 15, 25 and 79.     

Genotyping of Brucella melitensis and Brucella abortus strains currently circulating in Xinjiang,China

Brucellosis is a well-known zoonotic disease that can cause severe economic and healthcare losses. Xinjiang, one of the biggest livestock husbandry sectors in China, has gone through increasing incidence of brucellosis in cattle and small ruminants recently. In this paper, 50 B. melitensis strains and 9 B. abortus strains collected from across Xinjiang area (from 2010 to 2015) were genotyped using multiple locus variable-number tandem-repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST). Based on 8 loci (MLVA-8), 50 B. melitensis strains were classified into three genotypes. Genotypes 42 (n = 38, 76%) and 63 (n = 11, 22%) were part of the East Mediterranean group, and one genotype with pattern of 1-5-3-13-2-4-3-2 represents a single-locus variant from genotype 63. MLVA-16 resolved 50 B. melitensis strains into 28 genotypes, of which 15 are unique to Xinjiang and 10 are in common with those in adjacent country Kazakhstan and neighboring provinces of China. Minimum Spanning Tree (MST) analysis implies that B. melitensis strains collected from across Kazakhstan, Xinjiang and China areas may share a common origin. Nine B. abortus strains were sorted into three genotypes by MLVA-8, genotypes 36 (n = 7, 77.8%), 86 (n = 1, 11.1%) and a new genotype with pattern of 4-5-3-13-2-2-3-1. Each B. abortus strain showed distinct MLVA-16 genotypes, suggesting that B. abortus species may possess more genetic diversity than B. melitensis. Using MLST, most B. melitensis strains (n = 49) were identified as sequence type ST8, and most B. abortus strains (n = 8) were recognized as ST2. Two new sequence types, ST37 and ST38, represented by single strain from B. melitensis and B. abortus species respectively, were also detected in this study. These results could facilitate the pathogen surveillance in the forthcoming eradication programs and serve as a guide in source tracking in case of new outbreaks occur.     

Insights into the population structure of Mycobacterium tuberculosis using spoligotyping and RDRio in a southeastern Brazilian prison unit

Tuberculosis (TB) is still a serious public health problem, continuing to be an important threat for confined populations. We used spoligotyping to estimate the genotypic clades of Mycobacterium tuberculosis isolates from inmates in two blocks in a southeastern Brazilian prison unit, with TB incidence rate of 8185/100.000. The Latin American Mediterranean (LAM) clade is well represented in the country, and the LAM specific molecular markers, RD^Rio large sequence polymorphism and the SNP on the Rv3062 [ligB¹²¹²], were used to characterize spoligotype signatures from prison isolates. Typing of RD^Rio and ligB increase LAM clade from 66.7% (n = 72/108) to 69.4% (n = 75). The LAM2 SIT17 (n = 23) and SIT179 (n = 12) signatures comprised one third of all isolates, followed by Haarlem (11.5%, n = 12), T (8.7%, n = 9) and X (5.7%, n = 6) clades. Strains with unknown signatures represented 5.5% (n = 6), and four (3.7%) did not match any lineage. We observed RD^Rio among 64 (59.2%) isolates, and 54 (50%) were of the LAM clade. In particular, the LAM2/RD^Rio sub-lineage was significantly associated with clustering (p = 0.02) and its frequency was higher (32%) when compared to that of the previous general TB cases in RJ (4.29%). Overall cluster frequency defined by spoligotyping/IS6110-RFLP was 62%. The two evolutionary markers helped to evaluate some LAM signature misconceptions and demonstrate that LAM2/RD^Rio was found with high frequency, hitherto being unnoticed. All these data, allied to high clustering, imply that public health measures to minimize the escalation of TB in prison is essential, and both spoligotyping as well as RD^Rio would be useful tools to monitor the effects of the measures with respect to M. tuberculosis lineage variation.     

Global distribution and diversity of ovine-associated Staphylococcus aureus

Staphylococcus aureus is an important pathogen of many species, including sheep, and impacts on both human and animal health, animal welfare, and farm productivity. Here we present the widest global diversity study of ovine-associated S. aureus to date. We analysed 97 S. aureus isolates from sheep and sheep products from the UK, Turkey, France, Norway, Australia, Canada and the USA using multilocus sequence typing (MLST) and spa typing. These were compared with 196 sheep isolates from Europe (n = 153), Africa (n = 28), South America (n = 14) and Australia (n = 1); 172 bovine, 68 caprine and 433 human S. aureus profiles. Overall there were 59 STs and 87 spa types in the 293 ovine isolates; in the 97 new ovine isolates there were 22 STs and 37 spa types, including three novel MLST alleles, four novel STs and eight novel spa types. Three main CCs (CC133, CC522 and CC700) were detected in sheep and these contained 61% of all isolates. Four spa types (t002, t1534, t2678 and t3576) contained 31% of all isolates and were associated with CC5, CC522, CC133 and CC522 respectively. spa types were consistent with MLST CCs, only one spa type (t1403) was present in multiple CCs. The three main ovine CCs have different but overlapping patterns of geographical dissemination that appear to match the location and timing of sheep domestication and selection for meat and wool production. CC133, CC522 and CC700 remained ovine-associated following the inclusion of additional host species. Ovine isolates clustered separately from human and bovine isolates and those from sheep cheeses, but closely with caprine isolates. As with cattle isolates, patterns of clonal diversification of sheep isolates differ from humans, indicative of their relatively recent host-jump.     

Genotyping of Candida parapsilosis from three neonatal intensive care units (NICUs) using a panel of five multilocus microsatellite markers: Broad genetic diversity and a cluster of related strains in one NICU

Candida parapsilosis (CP) (n = 40) isolated from an unselected patient population in the neonatal intensive care units (NICUs) of three US hospitals were collected over periods of 3.5–9 years. Two previously published microsatellite markers and three additional trinucleotide markers were used to produce multiplex genotypes, which revealed broad strain diversity among the NICU isolates with a combined index of discrimination (D) = 0.997. A cluster of eight related CP strains from four infants in a single NICU was observed. An extended collection of 24 CP isolates from the general population of that hospital showed that the cluster of NICU isolates was related to three isolates from general hospital patients. This microsatellite marker set is suitable to investigate clusters of colonizing and infecting strains of CP.     

Common occurrence of Cryptosporidium hominis in horses and donkeys

Extensive genetic variation is observed within the genus Cryptosporidium and the distribution of Cryptosporidium species/genotypes in humans and animals appears to vary by geography and host species. To better understand the genetic diversity of Cryptosporidium spp. in horses and donkeys, we characterized five horse-derived and 82 donkey-derived Cryptosporidium isolates from five provinces or autonomous regions (Sichuan, Gansu, Henan, Inner Mongolia and Shandong) in China at the species/genotype and subtype levels. Three Cryptosporidium species/genotypes were identified based on the analysis of the SSU rRNA gene, including Cryptosporidium parvum (n = 22), the Cryptosporidium horse genotype (n = 4), and Cryptosporidium hominis (n = 61). The identification of C. hominis was confirmed by sequence analysis of the HSP70 and actin genes. Subtyping using sequence analysis of the 60 kDa glycoprotein gene identified 21 C. parvum isolates as subtype IIdA19G1, the four horse genotype isolates as subtypes VIaA15G4 (n = 2) and VIaA11G3 (n = 2), and the 61 C. hominis isolates as IkA16G1 (n = 59) and IkA16 (n = 2). The common finding of C. hominis reaffirms the heterogeneity of Cryptosporidium spp. in horses and donkeys and is possibly a reflection of endemic transmission of C. hominis in these animals. Data of the study suggest that horses and donkeys as companion animals may potentially transmit Cryptosporidium infections to humans.