Consequences of Long-term Exposure to Increased Salinity on the Amphibian Skin Bacterium Erwinia Toletana

Amphibian’s skin bacterial community may help them to cope with several types of environmental perturbations, including osmotic stress caused by increased salinity. This work aimed at assessing if an amphibian skin bacterium could increase its tolerance to NaCl after a long-term exposure to this salt. A strain of Erwinia toletana, isolated from the skin of Pelophylax perezi, was exposed to two salinity scenarios (of 18g/L of NaCl): (i) long-term exposure (for 46 days; Et-NaCl) and (ii) long-term exposure followed by a recovery period, (exposure for 30 days to NaCl and then to LB medium for 16 days; Et-R). After exposure, the sensitivity of E. toletana clonal populations to NaCl was assessed by testing 6 NaCl concentrations (LB medium spiked with NaCl) plus a control (LB medium). Genotypic alterations were assessed by PCR-based molecular typing method (BOX-PCR). Results shown that tolerance of E. toletana to NaCl slightly increased after the long-term exposure, EC50 for growth were: 22.5g/L (8.64-36.4) for Et-LB; 30.3g/L (23.2-37.4) for Et-NaCl, and 26.1g/L (19.3-32.9) for Et-R. Differences in metabolic activity were observed between Et-LB and Et-R and Et-NaCl and Et-R suggesting the use of different substrates by this bacterium when exposed to salinized environments. NaCl-induced genotypic alterations were not detected. This work suggests that E. toletana exposed to low levels of salinity, activate different metabolic pathways to cope with osmotic stress. Which may be further explored to be used in bioaugmentation procedures in natural populations of amphibians exposed to salinization.

A Core Genome Multilocus Sequence Typing Scheme for Streptococcus mutans

ABSTRACT Streptococcus mutans is one of the primary pathogens responsible for the development of dental caries. Recent whole-genome sequencing (WGS)-based core genome multilocus sequence typing (cgMLST) approaches have been employed in epidemiological studies of specific human pathogens. However, this approach has not been reported in studies of S. mutans. Here, we therefore developed a cgMLST scheme for S. mutans. We surveyed 199 available S. mutans genomes as a means of identifying cgMLST targets, developing a scheme that incorporated 594 targets from the S. mutans UA159 reference genome. Sixty-eight sequence types (STs) were identified in this cgMLST scheme (cgSTs) in 80 S. mutans isolates from 40 children that were sequenced in this study, compared to 35 STs identified by multilocus sequence typing (MLST). Fifty-six cgSTs (82.35%) were associated with a single isolate based on our cgMLST scheme, which is significantly higher than in the MLST scheme (11.43%). In addition, 58.06% of all MLST profiles with ≥2 isolates were further differentiated by our cgMLST scheme. Topological analyses of the maximum likelihood phylogenetic trees revealed that our cgMLST scheme was more reliable than the MLST scheme. A minimum spanning tree of 145 S. mutans isolates from 10 countries developed based upon the cgMLST scheme highlighted the diverse population structure of S. mutans. This cgMLST scheme thus offers a new molecular typing method suitable for evaluating the epidemiological distribution of this pathogen and has the potential to serve as a benchmark for future global studies of the epidemiological nature of dental caries. IMPORTANCE Streptococcus mutans is regarded as a major pathogen responsible for the onset of dental caries. S. mutans can transmit among people, especially within families. In this study, we established a new epidemiological approach to S. mutans classification. This approach can effectively differentiate among closely related isolates and offers superior reliability relative to that of the traditional MLST molecular typing method. As such, it has the potential to better support effective public health strategies centered around this bacterium that are aimed at preventing and treating dental caries.

Molecular Epidemiology of Streptococcus equi subsp. zooepidemicus Isolated from Thoroughbred Horses Using Multi Locus Sequence Typing (MLST) in Korea

Background:Multi Locus Sequence Typing (MLST) is a new global molecular typing method used for analyzing the DNA polymorphisms in bacteria. In this study, using MLST, we analyzed the sequence profiles ofStreptococcus (S.) zooepidemicusisolates from the Jeju and Jangsu provinces in South Korea.Objective:This study characterized the molecular epidemiology ofS. zooepidemicusinfection in Thoroughbred horses using MLST with an aim to control and prevent bacterial endometritis in mares.Methods:A total of 79S. zooepidemicusisolates were included in this study. Sequencing of the 7 loci for the MLST analysis was performed as described in the MLST website manual (http://pubmlst.org/szooepidemicus/) with some modifications. For each locus, every unique sequence was assigned a distinct allele number, and each Sequence Type (ST) was defined by a series of 7 integers (the allelic profile) corresponding to the alleles at the 7 loci (arcC,nrdE,proS,spi,tdk,tpi, andyqiL)using the MLST module in the Main Workbench.Results:Among the 79 isolates, 73 different MLST patterns were identified, each corresponding to 1-3 strains. The genetic relationships between the 79 isolates are presented in a dendrogram, and they diverged by up to 11% homology. At 11% homology, three MLST groups, M1, M2, and M3, were identified, and at 26% homology, five subgroups, L1-L5, were observed.We observed various MLST patterns in the strains isolated from Jeju and Jangsu. In addition, by estimating the epidemiological relationships among the strains isolated from Jangsu in 2007 and Jeju in 2009, which had similar MLST patterns, we determined that some strains from Jangsu may have been transported to Jeju.Conclusion:MLST can be used as a framework for determining the epidemiological relationships that form the genetic basis of host and infection site selection.

Utility of Whole Genome Sequencing To Describe the Persistence and Evolution of Listeria monocytogenes Strains within Crabmeat Processing Environments Linked to Two Outbreaks of Listeriosis

ABSTRACT This article describes the identification and investigation of two extended outbreaks of listeriosis in which crabmeat was identified as the vehicle of infection. Comparing contemporary and retrospective typing data of Listeria monocytogenes isolates from clinical cases and from food and food processing environments allowed the detection of cases going back several years. This information, combined with the analysis of routinely collected enhanced surveillance data, helped to direct the investigation and identify the vehicle of infection. Retrospective whole genome sequencing (WGS) analysis of isolates provided robust microbiological evidence of links between cases, foods, and the environments in which they were produced and demonstrated that for some cases and foods, identified by fluorescent amplified fragment length polymorphism, the molecular typing method in routine use at the time, were not part of the outbreak. WGS analysis also showed that the strains causing illness had persisted in two food production environments for many years and in one producer had evolved into two strains over a period of around 8 years. This article demonstrates the value of reviewing L. monocytogenes typing data from clinical cases together with that from foods as a means of identifying potential vehicles and sources of infection in outbreaks of listeriosis. It illustrates the importance of reviewing retrospective L. monocytogenes typing alongside enhanced surveillance data to characterize extended outbreaks and inform control measures. Also, this article highlights the advantages of WGS analysis for strain discrimination and clarification of evolutionary relationships that refine outbreak investigations and improve our understanding of L. monocytogenes in the food chain.

PFGE: a tool for examination of heterogeneity between the bacterial spot-causing xanthomonads of tomato plants in Bulgaria

Pulsed-field gel electrophoresis (PFGE) is a highly discriminative molecular typing method that is used for epidemiological studies and investigation of outbreaks caused by different pathogens, including phytopathogenic Xanthomonas species. Bacterial spot (BS) is the most common and one of the most destructive diseases of tomato and pepper plants in Bulgaria. Several Xanthomonas species are known to cause BS, but the global distribution and genetic diversity of these species are not well understood. A collection of 100 BS-causing strains, isolated during the period of 1985–2012 from different tomato cultivars and weeds associated with tomato production areas from 11 geographic regions in Bulgaria, were screened for genetic diversity by genomic DNA restriction with rare-cutting endonucleases (XbaI and SpeI) subsequently resolved by PFGE. Two haplotypes for Xanthomonas vesicatoria and one haplotype for Xanthomonas gardneri strains were found.

Clustering of Staphylococcus aureus bovine mastitis strains from regions of Central-Eastern Poland based on their biochemical and genetic characteristics

Staphylococcus aureusstrains were isolated from mastitic milk of cows with infected mammary glands. The animals were living in 12 different farms near Lublin, in Central-Eastern Poland. A biochemical identification method based on enzymatic assay was performed, followed by haemolytic and proteolytic tests. PCR-RFLP targeted on thegapgene allowed the genetic identification of strains at the species level and verified phenotypic identification results. A molecular typing method using triplex PCR was performed to recognize the genetic similarity of the analyzed strains. DNA microarray hybridization (StaphyType, Alere Technologies) was used for detection of antibiotic resistance and virulence associated markers. The results obtained indicate high genetic similarity in strains isolated from the same sites. High genetic similarities were also detected between strains isolated from cows from different farms of the same region. A slightly lower similarity was noted however, in strains from various regions indicating that the strains are herd specific and that the cow’s infections caused byS. aureuswere of a clonal character. In 21 representative isolates selected for DNA-microarray testing, only fosfomycin (fosB) and penicillin resistance markers (blaZ, blaI, blaR) were detected. The presence of genes coding for haemolysins (lukF, lukS, hlgA, hla, hld, hlb), proteases (aur, sspA, sspB, sspP), enterotoxins (entA, entD, entG, entI, entJ, entM, entN, entO, entR, entU, egc-cluster), adhesins (icaA, icaC, icaD, bbp, clfA, clfB, fib, fnbA, map, vwb) or immune evasion proteins (scn, chp, sak) was common and, with exceptions, matched triplex PCR-defined clusters.

Antimicrobal resistance ofNeisseria gonorrhoeaestrains in Hungary

Introduction: European guidelines on the treatment of Neisseria gonorrhoeae are based mostly on Western European data, although these recommendations may not be optimised for the circumstances in Hungary. Aim: The aim of the authors was to assess current antimicrobial resistance of Neisseria gonorrhoeae strains in order to enhance gonococcal antimicrobial surveillance in Hungary. Neisseria gonorrhoeae strains were isolated at the National Center of Sexually Transmitted Infections at the Department of Dermatology, Venerology and Dermatooncology of Semmelweis University in the period between January 2011 and June 2014. Method: Antimicrobial resistance was determined with minimum inhibitory concentration measurement. Neisseria gonorrhoeae Multiantigen Sequence typing was used as molecular typing method. Results: Resistance to the currently recommended extended spectrum cephalosporins is rare in Hungary, but there is an emerging azithromycin resistance among the Neisseria gonorrhoeae strains. Conclusions: Revision of the national treatment guideline must consider that the most frequent sequence types of Neisseria gonorrhoeae strains causing infections in Hungary are mainly resistant to azithromycin. Orv. Hetil., 2015, 156(6), 226–229.