Development and evaluation of a multi-locus sequence typing scheme for Mycoplasma synoviae

Reproducible molecular Mycoplasma synoviae typing techniques with sufficient discriminatory power may help to expand knowledge on its epidemiology and contribute to the improvement of control and eradication programmes of this mycoplasma species. The present study describes the development and validation of a novel multi-locus sequence typing (MLST) scheme for M. synoviae. Thirteen M. synoviae isolates originating from different poultry categories, farms and lesions, were subjected to whole genome sequencing. Their sequences were compared to that of M. synoviae reference strain MS53. A high number of single nucleotide polymorphisms (SNPs) indicating considerable genetic diversity were identified. SNPs were present in over 40 putative target genes for MLST of which five target genes were selected (nanA, uvrA, lepA, ruvB and ugpA) for the MLST scheme. This scheme was evaluated analysing 209 M. synoviae samples from different countries, categories of poultry, farms and lesions. Eleven clonal clusters and 76 different sequence types (STs) were obtained. Clustering occurred following geographical origin, supporting the hypothesis of regional population evolution. M. synoviae samples obtained from epidemiologically linked outbreaks often harboured the same ST. In contrast, multiple M. synoviae lineages were found in samples originating from swollen joints or oviducts from hens that produce eggs with eggshell apex abnormalities indicating that further research is needed to identify the genetic factors of M. synoviae that may explain its variations in tissue tropism and disease inducing potential. Furthermore, MLST proved to have a higher discriminatory power compared to variable lipoprotein and haemagglutinin A typing, which generated 50 different genotypes on the same database.     

O148 Shiga toxin-producing Escherichia coli outbreak: microbiological investigation as a useful complement to epidemiological investigation

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O148 infection occurred among wedding attendees in France in June 2002. A retrospective cohort study was performed and ten cases were identified, including two adults with haemolytic uraemic syndrome (HUS). The analytical study revealed that > 80% of affected individuals had eaten lightly roasted mutton and poultry paté, but only the consumption of paté tended to be associated with illness (relative risk 3.4; 95% CI 0.8-14.4). Left-overs (cooked mutton and raw offal) and processed foods (paté) from the same batches as served at the party were sampled. Human, food and environmental samples were examined for the Shiga toxin (stx) gene and virulence traits by PCR. Stx-positive samples were cultured for STEC. HUS cases were tested for serum antibodies against 26 major STEC serogroups. An STEC O26 strain (stx1, eae, ehxA) was isolated from one case with diarrhoea, and an STEC O148 strain (stx2c) from one case of HUS. Serum antibodies against O26 were not detected in either of these patients; antibodies against O148 were not tested. Three STEC strains were isolated from the mutton and the offal (stx2c, O148), and two from the paté (stx2c, O-X and O-Y). The isolates from the mutton were indistinguishable from the human stx2c isolate, whereas the paté isolates differed. Although four different STEC strains were identified in patients and foods, the results of molecular subtyping, in conjunction with analysis of food consumption patterns, strongly suggested that this outbreak was caused by mutton contaminated with STEC O148.     

Molecular Characterisation of Methicillin-Resistant Staphylococcus aureus Isolated from Patients at a Tertiary Care Hospital in Hyderabad,South India

Context: Infections with methicillin-resistant Staphylococcus aureus (MRSA) greatly influence clinical outcome. Molecular characterisation of MRSA can help to predict their spread and to institute treatment and hospital protocols. Aim: The aim of this study is to understand the diversity of MRSA in a tertiary care hospital in Hyderabad, India. Settings and Design: Samples collected at Gandhi Medical College, Hyderabad, and designed to assess hospital-or community-associated MRSA (HA-MRSA or CA-MRSA). Subjects and Methods: MRSA were subjected to antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE), spa typing, multi-locus sequence typing and staphylococcal cassette chromosome–mec (SCCmec) typing. Statistical Analysis Used: Discriminatory index and 95% confidence interval. Results: Of the 30 MRSA, (a) 18 and 12 were HA-MRSA and CA-MRSA, respectively, and (b) 23.3% and 6.6% displayed induced clindamycin and intermediate vancomycin resistance, respectively. Genetic diversity was evident from the presence of (a) 20 pulsotypes, (b) eight spa types, with the predominance of t064 (n = 9) and (c) seven sequence types (ST), with the preponderance of ST22 and ST8 (9 each). ST22 and ST8 were the most prevalent among HA-MRSA and CA-MRSA, respectively. SCCmec type IV was the most frequent (n = 8). 44.4% of HA-MRSA belonged to SCCmec IV and V, whereas 33.3% of CA-MRSA belonged to SCCmec I and III; 33.3% (5/15) of the isolates harbouring the pvl gene belonged to SCCmec IVC/H. Conclusions: ST8 was a dominant type along with other previously reported types ST22, ST239, and ST772 from India. The observations highlight the prevalence of genetically diverse clonal populations of MRSA, suggesting potential multiple origins.     

Hospital outbreak caused by a carbapenem-resistant strain of Acinetobacter baumannii: patient prognosis and risk-factors for colonisation and infection

Between October 2001 and August 2002, 30 hospital patients became infected or colonised by a multiresistant (including to carbapenems) epidemic strain of Acinetobacter baumannii (AbMR) in a hospital outbreak. This study analysed the risk-factors associated with acquisition of this epidemic strain and investigated the prognosis of patients infected by AbMR, with the aim of elucidating factors which lead to mortality. A case-control study of the acquisition of AbMR in patients infected or colonised in the hospital outbreak was performed. Independent risk-factors leading to death were studied by logistic regression analysis. Multivariate analysis of the risk-factors for colonisation/infection with AbMR revealed an independent association with the presence of an arterial catheter (OR, 1.13; 95% CI, 1.03-1.25) and administration of imipenem as monotherapy (OR, 11.12; 95% CI, 2.33-53.09). Multivariate analysis of the prognostic features leading to mortality revealed a significant association with hypotension or shock (OR, 24.63; 95% CI, 1.56-387.56) at the time of bacterial isolation.     

Molecular epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains in four Mediterranean countries with a multilocus sequence typing scheme

Thirty-five multidrug-resistant Acinetobacter baumannii strains, representative of 28 outbreaks involving 484 patients from 20 hospitals in Greece, Italy, Lebanon and Turkey from 1999 to 2009, were analysed by multilocus sequence typing. Sequence type (ST)2, ST1, ST25, ST78 and ST20 caused 12, four, three, three and two outbreaks involving 227, 93, 62, 62 and 31 patients, respectively. The genes bla_oxa-58, bla_oxa–23 and bla_oxa-72 were found in 27, two and one carbapenem-resistant strain, respectively. In conclusion, A. baumannii outbreaks were caused by the spread of a few strains.     

A nosocomial outbreak of KPC-2-producing Klebsiella pneumoniae in a Chinese hospital: dissemination of ST11 and emergence of ST37, ST392 and ST395

In China, Klebsiella pneumoniae carbapenemase (KPC) -producing K. pneumoniae isolates have been identified. However, little is known about the spread and outbreak of KPC-producing enterobacterial pathogens. In this study, 48 non-duplicated KPC-producing isolates were analysed for genetic relatedness by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility by E-test, and sequence type (ST) by multilocus sequence typing. S1-PFGE and Southern blot were used for plasmid profiling, and PCR and subsequent sequencing were performed to determine the effects of genetic background on the blaKPC gene. From December 2011 to June 2012, an outbreak of the KPC-2-producing K. pneumoniae was observed. The 48 isolates of K. pneumoniae are categorized into eight PFGE types (A1, A2, A3, A4, B, C, D and E). The predominant pathogens of the outbreak were strains with PFGE types A1, A2 and A3, which all belong to ST11. Furthermore, ST37, ST392 and ST395 KPC-2-producing K. pneumoniae isolates have also been sporadically identified. The blaKPC-2-carrying plasmids vary in size from 30 to 220 kb. The genetic environments of the blaKPC-2 gene for most strains were consistent with the genetic structure of blaKPC-2 on the plasmid pKP048. In conclusion, the dissemination and outbreak of KPC-2-producing K. pneumoniae isolates in this study appeared to be clonal, and ST11 K. pneumoniae was the predominant clone attributed to the outbreak. This is the first study to report the emergence and spread of KPC-producing K. pneumoniae ST392 and ST395 worldwide. Our findings suggest that horizontal transfer of Tn3-based transposons might mediate the spread of blaKPC-2 gene between different K. pneumoniae clones in China.     

Ultrasound as a Tool in Experimental Research of Macro- and Microcirculation

Peculiarities of application of two modes of ultrasonic measurements of blood flow in experimental research are considered, which are based on Doppler effect and on differential transit-time of upstream and downstream sound propagation. The efficiency of high-frequency ultrasound flowmeter equipped with 26.8 MHz transducers was demonstrated in measurements of blood flow in rat midbrain and coronary arteries. This approach can be used for evaluation of the dynamics of cardiac output with an intravascular catheter 0.6 mm in diameter working at 33 MHz. The probe and electronic scheme of the devise for measuring blood flow in microvessels are described. Blood flow rate measured in mesentery and m. cremaster arterioles under normal conditions was 2-12 mm/sec. One-element probe working at 38.5 MHz provided stable recording of blood microflows in 30-40- vessels.     

Genetic relatedness between group B streptococci originating from bovine mastitis and a human group B streptococcus type V cluster displaying an identical pulsed-field gel electrophoresis pattern

Twenty isolates of group B streptococcus (GBS) were recovered from the milk of cows with bovine mastitis on three farms located in the south and south-east of Brazil between 1987 and 1988. These isolates were characterised by molecular methods and compared with a collection of 103 human GBS isolates from colonised and infected patients in the same region between 1980 and 2003. Some of the bovine isolates shared identical or similar pulsed-field gel electrophoresis (PFGE) patterns with a PFGE clone of human GBS type V. In addition, these bovine and human isolates also possessed the same ribotype. Multilocus sequence typing (MLST) of representative isolates confirmed the genetic relationship between the human and bovine GBS isolates with identical PFGE patterns, which clustered in the same ST-26 clonal complex. These data support the hypothesis that some bovine GBS strains are related closely to human isolates and may infect humans, or vice versa. Further comparative genomic analyses of GBS isolates from bovine and human origins are required to investigate this hypothesis further.     

Characterisation of Yersinia pestis isolates from natural foci of plague in the Republic of Georgia, and their relationship to Y. pestis isolates from other countries

Forty Yersinia pestis isolates from endemic foci of plague in the Republic of Georgia, and six Y. pestis isolates from neighbouring former Soviet Union countries, were analysed for their biochemical and phenotypic properties, and their genetic relatedness was compared with Y. pestis strains KIM and CO92 by pulsed-field gel electrophoresis (PFGE). In addition, 11 Y. pestis isolates from the USA, together with published nucleotide sequences from Y. pestis strains KIM, CO92 and 91001, were compared with the 46 isolates in the present collection using multilocus sequence typing (MLST), based on sequence data for the 16S rRNA, hsp60, glnA, gyrB, recA, manB, thrA and tmk loci. Four virulence gene loci (caf1, lcrV, psaA and pla) were also sequenced and analysed. Two sequence types (ST1 and ST2), which differed by a single nucleotide, were identified by MLST. With the exception of a single isolate (771G), all of the Georgian Y. pestis isolates belonged to ST2. PFGE also grouped the Georgian Y. pestis isolates separately from the non-Georgian isolates. Overall, PFGE discriminated the Y. pestis isolates more effectively than MLST. The sequences of three of the four virulence genes (lcrV, psaA and pla) were identical in all Georgian and non-Georgian isolates, but the caf1 locus was represented by two allele types, with caf1 NT1 being associated with the non-Georgian isolates and caf1 NT2 being associated with the Georgian isolates. These results suggest that Georgian Y. pestis isolates are of clonal origin.     

Molecular identification of adenovirus sequences: a rapid scheme for early typing of human adenoviruses in diagnostic samples of immunocompetent and immunodeficient patients

Precise typing of human adenoviruses (HAdV) is fundamental for epidemiology and the detection of infection chains. As only few of the 51 adenovirus types are associated with life- threatening disseminated diseases in immunodeficient patients, detection of one of these types may have prognostic value and lead to immediate therapeutic intervention. A recently published molecular typing scheme consisting of two steps (sequencing of a generic PCR product closely adjacent to loop 1 of the main neutralization determinant epsilon, and for species HAdV-B, -C, and -D the sequencing of loop 2 [Madisch et al., 2005]) was applied to 119 clinical samples. HAdV DNA was typed unequivocally even in cases of culture negative samples, for example in immunodeficient patients before HAdV causes high virus loads and disseminated disease. Direct typing results demonstrated the predominance of HAdV-1, -2, -5, and -31 in immunodeficient patients suggesting the significance of the persistence of these viruses for the pathogenesis of disseminated disease. In contrast, HAdV-3 predominated in immunocompetent patients and cocirculation of four subtypes was demonstrated. Typing of samples from a conjunctivitis outbreak in multiple military barracks demonstrated various HAdV types (2, 4, 8, 19) and not the suspected unique adenovirus etiology. This suggests that our molecular typing scheme will be also useful for epidemiological investigations. In conclusion, our two-step molecular typing system will permit the precise and rapid typing of clinical HAdV isolates and even of HAdV DNA in clinical samples without the need of time-consuming virus isolation prior to typing.     

Emergence and control of an outbreak of infections due to Panton-Valentine leukocidin positive,ST22 methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit

Methicillin resistant Staphylococcus aureus (MRSA) infection can cause significant morbidity and mortality in neonates. We investigated a nosocomial MRSA outbreak in a neonatal intensive care unit (NICU), using a novel typing method. Following two fatal cases, in May 2011, a prospective outbreak investigation was conducted, involving neonates, mothers and healthcare workers in a large tertiary NICU in Sydney. MRSA isolates were characterized by antimicrobial susceptibility testing, a multiplex PCR-based reverse line blot (mPCR/RLB) binary typing system and other molecular typing methods. Over 7 months, 14 neonates were colonized with MRSA and six infected: three with superficial lesions and three with life-threatening disease, including the two index cases, who died despite empirical treatment with vancomycin. Isolates from 15 neonates were indistinguishable by RLB typing and identified as a PVL-producing ST22 SCCmec IV MRSA strain, which was resistant to gentamicin and trimethoprim-sulphamethoxazole. The outbreak strain was also isolated from one healthcare worker, one environmental swab and one father, but the source remained obscure. During the same period several different non-multiresistant and multiresistant MRSA strains were isolated from five neonates, five mothers (including two whose infants were colonized with the outbreak strain), one father, three healthcare workers and two environmental swabs. Rapid turnaround time of typing results allowed us to recognize and define the outbreak and implement targeted infection control interventions. PVL-producing ST22 SCCmec IV MRSA appears to be a virulent and highly transmissible pathogen in the NICU, which was difficult to control.     

Determination of Amniotic Fluid IgM and IgA by Hydroxyethylcellulose-Agarose Gel Electroimmunodiffusion

ABSTRACT: An electroimmunodiffusion method on hydroxyethylcellulose-agarose by which IgA and IgM concentrations down to 0.3 mg/dl can be measured in amniotic fluid is presented. Elevated Ig levels were found in both fetal infection and in congenital malformations. The potential value of Ig determination in amniotic fluid is discussed.     

Genetic Diversity and Allelic Variation in South Indian Isolates of Group A Streptococci Causing Invasive Disease

Background: Reported literature on invasive group A streptococcal isolates in India is very scanty. This study was undertaken to determine the molecular heterogeneity of such isolates as seen in a tertiary care center. Materials and Methods: Thirty two blood culture isolates and 18 from other sterile body fluids were characterized by emm gene sequencing and multilocus sequence typing. Results: Forty two emm types were identified including 25 from 32 blood isolates and 17 from 18 other body fluid isolates. Types 110, 74, 63, 85, 102, 105, 124 and st854.1 were common to both groups and accounted for 40% of the isolates. Two types namely, stKNB6 and stKNB9 were newly identified types. MLST identified forty eight sequence types (MLST - ST) of which 31 were from 32 blood isolates and 17 from 18 body fluid isolates; thirty three of them were hitherto unrecognized at the time of identification. Two blood isolates of emm 85 had the same MLST - ST 484 while three blood isolates of emm 110 had three different STs namely, ST 493, 494 and 497. Two types, ST 493 and ST497 had single locus variation while ST 497 had a double locus variation. Conclusions: Our study shows that subtle allelic variations in the house keeping genes results in the development of new strains in a given emm type and contribute significantly to the existing high diversity of strains circulating in the community.     

The ORION guidelines: raising research standards for the control of antimicrobial-resistant pathogens and healthcare-associated infection

The ORION (Outbreak Reports and Intervention studies Of Nosocomial Infection) guidelines were recently published with the aim of raising methodological standards and the clarity of reporting of intervention studies in hospital infection control. The guidelines provide advice on use of appropriate statistical analysis techniques and the measures necessary to prevent bias, with the aim of ensuring complete transparency in reports of such interventions, their epidemiological context and potential confounders. CMI authors and reviewers should consider these helpful proposals when designing, reporting and assessing intervention and outbreak studies. Use of these guidelines should contribute to the construction of a solid evidence base for control of antimicrobial resistance and healthcare-associated infection.     

Identification of a novel MICA allele: MICA*051

We have identified a novel MICA allele, MICA*051, detected by the polymerase chain reaction using sequence-specific primers and characterized by sequence-based typing. MICA*051 appears to be the result of a recombination between MICA*00801 and MICA*00701 at intron 2.     

USA300 abroad: global spread of a virulent strain of community-associated methicillin-resistant Staphylococcus aureus

The epidemic of USA300-0114 methicillin-resistant Staphylococcus aureus (MRSA) in the USA has been remarkable for its virulence and for its ability to cause infections in both the community and healthcare settings. Although it has mainly been associated with skin and soft tissue infections, particularly furunculosis, it has also caused severe life-threatening conditions such as necrotizing pneumonia, osteomyelitis, and septic arthritis. This strain or a closely related Latin American variant has now spread to multiple countries on five continents, where associated clinical and epidemiological features have been in keeping with those seen in the USA. Furthermore, it has become the dominant community-associated MRSA strain in five countries. It is now a major international epidemic strain, but whether it will supplant established community-associated strains in other countries remains to be seen.     

Molecular epidemiology of clinical Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates using a multilocus sequencing typing scheme

To further expand the limited multilocus sequence typing (MLST) database for Acinetobacter baumannii , 53 clinical isolates from various outbreaks in Europe and the USA, collected between 1991 and 2004, plus the A. baumannii reference strain ATCC 19606^T and 20 clinical Acinetobacter genomic species 13TU isolates from the same period, were analyzed using a new MLST scheme based on fragments of the gltA , gyrB , gdhB , recA , cpn60 , gpi and rpoD genes. Data were compared with typing results generated using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD)-PCR. In total, 50 sequence types (STs) were distinguished among the A. baumannii isolates investigated, and the MLST data were in high concordance with the PFGE and RAPD-PCR results. Only five clonal complexes were identified by eBURST analysis, including the 21 STs listed in a previous study, suggesting high diversity among the A. baumannii isolates. With one exception, there was no relatedness among isolates from outbreaks in different countries (Europe) or regions (USA). No intercontinental spread was revealed. Acinetobacter genomic species 13TU isolates could also be analyzed using the A. baumannii MLST scheme (18 different STs) and could be distinguished from A. baumannii isolates according to characteristic sequences. It was concluded that the MLST scheme provides a high level of resolution and is a promising tool for studying the epidemiology of A. baumannii and Acinetobacter genomic species 13TU.