Dissemination of a 'rare' extended-spectrum ß-lactamase gene blaSFO-1 mediated by epidemic clones of carbapenemase-producing Enterobacter hormaechei in China.

An increasing trend of the coexistence of a rare extended-spectrum ß-lactamase gene blaSFO-1 and carbapenemase genes in Enterobacteriaceae has recently been noted. This study aimed to determine the epidemiological and genetic characterisation of SFO-1-positive carbapenem-resistant Enterobacter cloacae complex (CREC) isolates. A total of 61 CREC clinical isolates were collected in the framework of a national surveillance for carbapenem-resistant Enterobacteriaceae during 2011-2015 in China. Seven SFO-1-positive CREC isolates (11.5%) were identified in four provinces, suggesting a wide dissemination of the blaSFO-1 gene among the CREC population in China. Five SFO-1-positive CREC isolates were further identified by screening 1625 genomes of E. cloacae complex strains retrieved from GenBank. The 12 SFO-1-positive CREC isolates were further identified as Enterobacter hormaechei, of which 10 belonged to epidemic clones (ST93, ST114 and ST418), indicating that these clones might largely contribute to the dissemination of blaSFO-1. Phylogenomics analysis further identified the occurrence of clonal dissemination in the community setting. The blaSFO-1-bearing plasmids were assigned to various incompatibility groups with highly diverse sizes (~104-370 kb), suggesting a wide vector range of blaSFO-1. Two types of genetic context, with and without insertion sequence IS26, were identified for the blaSFO-1 gene. The genetic context flanked by IS26 was more prevalent, thus largely facilitating the mobility of blaSFO-1. This study revealed that the blaSFO-1 gene is not as rare as previously found and that epidemic clones of CREC are responsible for its dissemination in China. These findings highlight the potential of wide dissemination of low-prevalence antimicrobial resistance genes.

Isolation of Candida auris from invasive and non-invasive samples of a patient suffering from vascular disease, Italy, July 2019.

We recently isolated Candida auris from a blood culture and cutaneous swabs of a patient in her mid-70s. Our routine phenotypic methods failed to identify the microorganism, but it was identified by molecular tests and MALDI-TOF MS analysis. Our report, the first from Italy, further underlines the geographically wide distribution of C. auris and the need to confirm species identification of any suspicious colony as soon as possible to stop its spread.

Prevalence, Genotypic Characteristics and Antibiotic Resistance of Listeria monocytogenes From Retail Foods in Bulk in Zhejiang Province, China.

Listeria monocytogenes is an important foodborne pathogen causing public concern. A total of 3354 retail foods in bulk were sampled and screened for L. monocytogenes. Seventy-three (2.2%) samples including 21 ready-to-eat (RTE) foods and 52 raw foods were confirmed positive for L. monocytogenes. Sushi and salmon sashimi occupied the top two slots in RTE foods with relatively high presence rate of 12.9 and 6.9%, respectively. Meanwhile, L. monocytogenes was found to be distributed unequally in raw foods; the presence rates in raw meat (3.5%) and poultry (3.8%) were significantly higher than that in raw seafood (1.3%). Notably, L. monocytogenes was not detected in raw freshwater food. The L. monocytogenes isolates belonged to four serotypes, 1/2a, 1/2b, 1/2c, and 4b, with the most prevalent serotype being 1/2a (47.9%). Eighteen sequence types (STs) and eighteen virulence types (VTs) containing four newly assigned VTs (VT180, VT181, VT182, and VT183) were determined via multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST). Among the 73 L. monocytogenes isolates, 23 (31.5%) belonged to epidemic clones (ECs) including ECI, ECIV, ECV, ECVI, ECVIII and ECXI among which ECV was predominant. Antibiotic susceptibility tests revealed a high resistance rate (11.0%) to tetracycline. Moreover, we identified the distribution patterns of virulence genes of four Listeria pathogenicity islands (LIPI) in L. monocytogenes isolates. prfA, hly, plcA, plcB, mpl, actA genes in LIPI-1 and inlA, inlB, inlC, inlJ genes in LIPI-2 were detected in approximately all L. monocytogenes isolates. The distribution of both LIPI-3 genes and LIPI-4 genes exhibited association with lineage and ST. LIPI-4 genes were present exclusively in ST87 isolates. Relatedness analysis revealed the absence of distinct association between STs, ECs, LIPI-3 and LIPI-4 distribution and specific food groups. This study provided fundamental data for Chinese food safety authorities to grasp the contamination status of L. monocytogenes in foods, assess the potential risk of this pathogen and further address the safety issue of retail foods in bulk in China.

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination.

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations.

Identification of a major Listeria monocytogenes outbreak clone linked to soft cheese in Northern Italy - 2009-2011.

BACKGROUND: Molecular subtyping and enhanced surveillance in Lombardy region identified a cluster of possibly related listeriosis cases from 2006 to 2010. This cluster grouped 31 isolates that belonged to serotype 1/2a and Sequence Type 38 (ST38) as defined by Multilocus Sequence Typing (MLST). METHODS: Our study expanded the previous investigation to include cases from 2011 to 2014 and used Multi-Virulence-Locus Sequence Typing (MVLST) on all ST38 isolates to better understand their epidemiology and possibly identify a common source outbreak. RESULTS: Out of 306 L. monocytogenes clinical isolates collected, 43 (14.1%) belonged to ST38 with cases occurring in nine out of twelve Lombardy provinces. The ST38 isolates were split by MVLST into two Virulence Types (VTs): VT80 (n = 12) and VT104 (n = 31). VT104 cases were concentrated between 2009 and 2011 in two provinces, Bergamo and Milan. An epidemiologic investigation was performed and in one case, a matching VT104 isolate was retrieved from a soft cheese sample from a patient's refrigerator. CONCLUSIONS: Our findings revealed a major listeriosis outbreak in Northern Italy linked to soft cheese in 2009-2011, which went undetected by local health authorities. Our study shows that integrating subtyping methods with conventional epidemiology can help identify the source of L. monocytogenes outbreak clones.

A national survey of the molecular epidemiology of Clostridium difficile in Israel: the dissemination of the ribotype 027 strain with reduced susceptibility to vancomycin and metronidazole.

Our goals were to study the molecular epidemiology and antimicrobial susceptibilities of C. difficile strains in Israel. Microbiology laboratories serving 6 general hospitals (GH) and 10 long-term care facilities (LTCF) were asked to submit all stool samples in January-February 2014 that tested positive for C. difficile. Toxigenic C. difficile isolates were recovered in 208 out of 217 samples (95.8%), of which 50 (23.6%) were from LTCFs. Ribotype 027 was the most common type overall, identified in 65 samples (31.8%), and was the predominant strain in the 3 GHs with the highest incidence of C. difficile infections. Other common strains were slpA types cr-02 (n = 45) and hr-02 (n = 18). The proportions of vancomycin and metronidazole MIC values >2mg/L were high in ribotype 027 (87.7% and 44.6%, respectively) and slpA-cr-02 strains (88.8% and 17.8%, respectively). This study demonstrates that the ribotype 027 strain has disseminated across Israel and is now the most common strain.

Biofilm production by multiresistant Corynebacterium striatum associated with nosocomial outbreak

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum 1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatum bound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

Development and validation of a multiplex PCR assay for identification of the epidemic ST-258/512 KPC-producing Klebsiella pneumoniae clone.

The Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-KP) sequence type (ST)-258/512 clone is the dominant clone by which KPC has disseminated worldwide. Standard typing methods are time-consuming and are therefore impractical for identification of this clone in the course of an outbreak. Through comparative genomic study, we have previously identified several presumably unique genes of this clone: 1) PILV-like protein (pilv-l), 2) transposase, IS66-family (is-66), and a 3) phage-related protein (prp). Our aims were to 1) test for the presence of these genes using a multiplex PCR in a large, multinational collection of KPC-KP isolates and to 2) validate this assay as a typing method for the identification of the ST-258/512 clone. KPC-KP isolates (n=160) that included both ST-258/512 (group A, n=114) and non-ST-258 (group B, n=46) strains were collected from the following countries: Greece, 20; Israel, 93; Italy, 19; USA, 25; and Colombia, 3. Group B included 30 different STs from various lineages. The pilv-l gene was present in 111/114 of ST-258 isolates, including all of the KPC-negative isolates resulting in a sensitivity of 97%. Using primers for a unique ST-258 pilv-l allele resulted in a specificity of 100%. The sensitivity values of is-66 and prp genes for detecting KPC-KP ST-258 were 83 and 89%, respectively, and the specificity values were 67 and 93%, respectively. PCR for the unique pilv-l ST-258 allele provides a reliable tool for rapid detection of the ST-258 clone. This method can be helpful both in the setting of an outbreak and in a large-scale survey of KPC-KP strains.

Acinetobacter baumannii: evolution of a global pathogen.

Acinetobacter baumannii is an opportunistic nosocomial pathogen and one of the six most important multidrug-resistant microorganisms in hospitals worldwide. This human pathogen is responsible for a vast array of infections, of which ventilator-associated pneumonia and bloodstream infections are the most common, and mortality rates can reach 35%. Community-acquired infections have also been reported, but few strains have been recovered from environmental sources and infection reservoirs external to the hospital have not been identified. The majority of A. baumannii infections are caused by two main population clones with worldwide distribution. Infection outbreaks are often associated with multidrug resistance, including the recent emergence of strains resistant to all available antibiotics. Nevertheless, A. baumannii virulence traits and pathogenic potential have mostly remained elusive. The recent expansion of A. baumannii sequenced genomes has permitted the development of large-array phylogenomic and phenotypic analyses, which can offer valuable insights into the evolution and adaptation of A. baumannii as a human pathogen. This review summarises these recent advances, with particular focus on A. baumannii evolutionary and genomic aspects, and proposes new avenues of research.

Lagooning of wastewaters favors dissemination of clinically relevant Pseudomonas aeruginosa.

The significance of wastewater treatment lagoons (WWTLs) as point sources of clinically relevant Pseudomonas aeruginosa that can disseminate through rural and peri-urban catchments was investigated. A panel of P. aeruginosa strains collected over three years from WWTLs and community-acquired infections was compared by pulsed field gel electrophoresis (PFGE) DNA fingerprinting and multilocus sequence typing (MLST). Forty-four distantly related PFGE profiles and four clonal complexes were found among the WWTL strains analyzed. Some genotypes were repeatedly detected from different parts of WWTLs, including the influent, suggesting an ability to migrate and persist over time. MLST showed all investigated lineages to match sequence types described in other countries and strains from major clinical clones such as PA14 of ST253 and "C" of ST17 were observed. Some of these genotypes matched isolates from community-acquired infections recorded in the WWTL geographic area. Most WWTL strains harbored the main P. aeruginosa virulence genes; 13% harbored exoU-encoded cytoxins, but on at least six different genomic islands, with some of these showing signs of genomic instability. P. aeruginosa appeared to be highly successful opportunistic colonizers of WWTLs. Lagooning of wastewaters was found to favor dissemination of clinically relevant P. aeruginosa among peri-urban watersheds.

Molecular approaches to the identification of pathogenic and nonpathogenic listeriae.

The genus Listeria consists of a closely related group of Gram-positive bacteria that commonly occur in the environment and demonstrate varied pathogenic potential. Of the 10 species identified to date, L. monocytogenes is a facultative intracellular pathogen of both humans and animals, L. ivanovii mainly infects ungulates (eg., sheep and cattle), while other species (L. innocua, L. seeligeri, L. welshimeri, L. grayi, L. marthii, L. rocourtiae, L. fleischmannii and L. weihenstephanensis) are essentially saprophytes. Within the species of L. monocytogenes, several serovars (e.g., 4b, 1/2a, 1/2b and 1/2c) are highly pathogenic and account for a majority of clinical isolations. Due to their close morphological, biological, biochemical and genetic similarities, laboratory identification of pathogenic and nonpathogenic Listeria organisms is technically challenging. With the development and application of various molecular approaches, accurate and rapid discrimination of pathogenic and nonpathogenic Listeria organisms, as well as pathogenic and nonpathogenic L. monocytogenes strains, has become possible.

Antimicrobial activity and bioautographic study of antistaphylococcal components from Caesalpinia pyramidalis Tull

The antimicrobial activity of dry methanol and ethyl acetate extracts for the leaves, bark of the stem, peel of the root, flower, fruit and seed of Caesalpinia pyramidalis Tull. (catingueira) was performed against seventeen isolates of Staphylococcus aureus MRSA multiresistant strains, which included two isolates of S. aureus MSSA and two ATCC strains. The antimicrobial activity was tested by the agar diffusion method and the Minimum Inhibitory Concentration (MIC) was determined. The dry methanol extract of the root showed good antimicrobial activity with a MIC of less than 0.5 mg.mL-1. The dry ethyl acetate extracts exhibited lower antimicrobial activity, which might be explained by solubility problems and less diffusion in the agar medium. Results of the bioautographies also confirmed inhibition halos corresponding to the active substances present in the leaves, as well as in the flower of C. pyramidalis. The phytochemical study of the leaves, bark of the stem, peel of the root, flower and fruit of extracts from C. pyramidalis confirmed the presence of a number of known antimicrobial agents including ursolic acid, quercetin, catechin, ellagic acid, sitosterol, flavonoids, proanthocyanidins and gallic acid.

A determinação da atividade antimicrobiana dos extratos metanólicos e em acetato de etila da folha, casca do caule, casca da raiz, flor, fruto e semente de Caesalpinia pyramidalis Tull. foi realizada frente a dezessete isolados de Staphylococcus aureus MRSA multirresistentes, dois isolados de S. aureus MSSA e duas cepas padrão, pelas técnicas de poço/difusão em ágar e determinação das CMI pelo método de diluição em agar/multiinoculador de Stears. O extrato metanólico de casca da raiz indicou uma boa atividade, com CMI inferior a 0.5 mg.mL-1. Os extratos secos por extração em acetato de etila apresentaram menor atividade que se poderia explicar por problemas de solubilidade e menor difusão no meio de cultura em ágar. Resultados das bioautografias confirmaram zonas de inibição correspondente às substâncias ativas presente na folha, como também na flor da C. pyramidalis. No estudo fitoquímico das folhas, casca da caule, casca da raiz, flor e fruto dos extratos de C. pyramidalis evidenciou-se a presença de vários constituintes com reconhecida atividade antimicrobiana, entre estes o ácido ursólico, quercetina, catequina, ácido elágico, sitosterol, flavonóides, proantocianidinas e ácido gálico. Entre todos os metabólitos citados, somente o ultimo não observamos, por CCD, na casca da raiz de C. pyramidalis.

The current role of pulsed-field gel electrophoresis in methicillin-resistant Staphylococcus aureus (MRSA) typing and the retrospective identification of outbreaks.

The objective of this paper was to investigate whether retrospective pulsed-field gel electrophoresis (PFGE) of methicillin-resistant Staphylococcus aureus (MRSA) isolates at two-year intervals is suitable and sufficient to demonstrate changes in the clonal composition of MRSA isolates and to identify previously undetected local outbreaks. PFGE patterns of 400 MRSA isolates were collected between 2004 and 2008 at the University of Rostock Hospital in Germany, and were used to assess the prevalence of MRSA clones at different time points. Only minor changes were detected. The combined analysis of all isolates that were collected per year reduced the time needed to perform this laborious procedure. The retrospective identification of outbreaks may require shorter intervals. Improved infection prevention and control measures prevented further outbreaks in previously affected hospital departments. In conclusion, PGFE at two-year intervals is sufficient to detect changes in the clonal composition of local MRSA isolates. If time for identification is important during outbreak investigations, more rapid methods with a similarly high discriminatory power such as spa typing should be used.

Genotyping of methicillin-resistant Staphylococcus aureus isolates obtained in the Northeast region of Brazil

Methicillin-resistant Staphylococcus aureus (MRSA) is a major agent of hospital infections worldwide. In Brazil, a multiresistant MRSA lineage (ST239-SCCmecIIIA), the so-called Brazilian epidemic clone (BEC), has predominated in all regions. However, an increase in nosocomial infections caused by non-multiresistant MRSA clones has recently been observed. In the present study, 45 clinical isolates of MRSA obtained from a university hospital located in Natal city, Brazil, were identified by standard laboratory methods and molecularly characterized using staphylococcal chromosome cassette mec (SCCmec) typing and pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was carried out using CLSI methods. The MRSA isolates studied displayed a total of 8 different pulsed-field gel electrophoresis patterns (types A to H) with predominance (73 percent) of pattern A (BEC-related). However, MRSA harboring SCCmec type IV were also identified, 3 (7 percent) of which were genetically related to the pediatric clone - USA800 (ST5-SCCmecIV). In addition, we found a considerable genetic diversity within BEC isolates. MRSA displaying SCCmecIV are frequently susceptible to the majority of non-β-lactam antibiotics. However, emergence of multiresistant variants of USA800 was detected.