Increased prevalence of indoor Aspergillus and Penicillium species is associated with indoor flooding and coastal proximity: a case study of 28 moldy buildings.

Indoor flooding is a leading contributor to indoor dampness and the associated mold infestations in the coastal United States. Whether the prevalent mold genera that infest the coastal flood-prone buildings are different from those not flood-prone is unknown. In the current case study of 28 mold-infested buildings across the U.S. east coast, we surprisingly noted a trend of higher prevalence of indoor Aspergillus and Penicillium genera (denoted here as Asp-Pen) in buildings with previous flooding history. Hence, we sought to determine the possibility of a potential statistically significant association between indoor Asp-Pen prevalence and three building-related variables: (i) indoor flooding history, (ii) geographical location, and (iii) the building's use (residential versus non-residential). Culturable spores and hyphal fragments in indoor air were collected using the settle-plate method, and corresponding genera were confirmed using phylogenetic analysis of their ITS sequence (the fungal barcode). Analysis of variance (ANOVA) using Generalized linear model procedure (GLM) showed that Asp-Pen prevalence is significantly associated with indoor flooding as well as coastal proximity. To address the small sample size, a multivariate decision tree analysis was conducted, which ranked indoor flooding history as the strongest determinant of Asp-Pen prevalence, followed by geographical location and the building's use.

Incidence of Listeria monocytogenes and Listeria spp. in a small-scale mushroom production facility.

Listeria monocytogenes is a foodborne pathogen of significant concern to the agricultural and food processing industry because of its ability to grow and persist in cool and moist environments and its association with listeriosis, a disease with a very high mortality rate. Although there have been no listeriosis outbreaks attributed to fresh mushrooms in the United States, retail surveys and recalls are evidence that L. monocytogenes contamination of mushrooms (Agaricus bisporus) can occur. The objective of this study was to determine the prevalence of Listeria spp., including L. monocytogenes, in a small-scale mushroom production facility on the campus of the Pennsylvania State University in the United States. Of 184 samples taken from five production zones within the facility, 29 (15.8%) samples were positive for Listeria spp. Among the Listeria spp. isolates, L. innocua was most prevalent (10.3%) followed by L. welshimeri (3.3%), L. monocytogenes (1.6%), and L. grayi (0.5%). L. monocytogenes was recovered only from the phase I raw material composting area. Isolates of L. monocytogenes were confirmed and serotyped by multiplex PCR. The epidemiological relatedness of the three L. monocytogenes isolates to those serotypes or lineages frequently encountered in listeriosis infections was determined by multi-virulence-locus sequence typing using six virulence genes, namely, prfA, inlB, inlC, dal, clpP, and lisR. The phylogenetic positions of the three isolates in the dendrogram prepared with data from other isolates of L. monocytogenes showed that all isolates were grouped with serotype 4a, lineage IIIA. To date, this serotype has rarely been reported in foodborne disease outbreaks.

Novel epidemic clones of Listeria monocytogenes, United States, 2011.

We identified a novel serotype 1/2a outbreak strain and 2 novel epidemic clones of Listeria monocytogenes while investigating a foodborne outbreak of listeriosis associated with consumption of cantaloupe during 2011 in the United States. Comparative analyses of strains worldwide are essential to identification of novel outbreak strains and epidemic clones.

Klebsiella michiganensis sp. nov., a new bacterium isolated from a tooth brush holder.

Isolate W14(T) recovered from a household tooth brush holder was found to be gram-negative, a facultative anaerobic, non-motile, capsulated, and a non-endospore-forming straight rod. Based on phylogenetic analysis with 16S rRNA gene sequence, isolate W14(T) was affiliated to the genus Klebsiella. The closest phylogenetic relative was K. oxytoca with 99 % similarity in the 16S rRNA gene sequence. The major whole-cell fatty acids were C(16:0) (31.23 %), C(18:1ω6c)/C(18:1ω7c) (21.10 %), and C(16:1ω7c)/C(16:1ω6c) (19.05 %). The sequence similarities of isolate W14(T) based on rpoB, gyrA, and gyrB were 97, 98, and 98 % with K. oxytoca, and 97, 93, and 90 % with K. mobilis (=Enterobacter aerogenes), respectively. The ribotyping pattern showed a 0.46 similarity with K. oxytoca ATCC 13182(T) and 0.24 with K. mobilis ATCC 13048(T). The DNA G+C content of isolate W14(T) was 54.6 mol%. The DNA-DNA relatedness was 55.7 % with K. oxytoca ATCC 13182(T). Using the identification technology of MALDI-TOF mass spectrometry, the top matches for this isolate were K. oxytoca ATCC 13182(T) (Match Factor Score 1.998) and K. mobilis (Score 1.797). On the basis of phenotypic, biochemical, chemotaxonomic, and molecular studies, isolate W14(T) could be differentiated from other members of the genus Klebsiella including K. mobilis. Therefore, it is proposed that isolate W14(T) (=ATCC BAA-2403(T)=DSM 25444(T)) should be classified as the type strain of a novel species of the genus Klebsiella, K. michiganensis sp. nov.

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.

Sequence typing confirms that a predominant Listeria monocytogenes clone caused human listeriosis cases and outbreaks in Canada from 1988 to 2010.

Human listeriosis outbreaks in Canada have been predominantly caused by serotype 1/2a isolates with highly similar pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST) each identified a diverse population of Listeria monocytogenes isolates, and within that, both methods had congruent subtypes that substantiated a predominant clone (clonal complex 8; virulence type 59; proposed epidemic clone 5 [ECV]) that has been causing human illness across Canada for more than 2 decades.

Molecular evolutionary and epidemiological dynamics of a highly pathogenic fish rhabdovirus, the spring viremia of carp virus (SVCV).

Spring viremia of carp virus (SVCV), which causes an acute hemorrhagic and highly contagious disease in cyprinids, was first described in Europe and subsequently reported in parts of Asia and North America. SVCV can be classified into four genogroups: Ia, Ib, Ic, and Id. While Ia and Id have wide circulation and are reported to cause outbreaks in North America and Europe, respectively, Ib and Ic were last reported in the 1980s. We used a Bayesian framework to determine the nucleotide substitution rates, relative genetic diversity, and time to the most recent common ancestor (TMRCA) of SVCV using large sets of sequences of the phosphoprotein and glycoprotein (G) genes. The sampled genetic diversities of Ia and Id were found to have arisen during the year 1996 (95% Highest Posterior Density: 1986-1998) and 1957 (1926-1972), respectively, with consistent results across the two genes. The TMRCA for SVCV was estimated to have been around 1850 (1727-1938). The substitution rate for Ia is at least 5-7 times higher than that of Id. The rate of nonsynonymous (dn) to synonymous (ds) substitutions (dn/ds=ω) for the G gene of Ia (ω=0.608) is significantly higher than that of Id (ω=0.0749), indicating both exhibit distinct selection profiles. The SVCV population experienced a bottleneck during the early 1990 s followed by a sudden rebound, primarily due to the sudden increase in genetic variants in Ia and Id, which coincided with the timing of a recent series of outbreaks reported in Europe and North America.

A novel multiplex PCR method for detecting the major clonal complexes of MRSA in nasal isolates from a Pennsylvania hospital.

A novel multiplex PCR was developed which targeted virulence genes associated with the major clonal complexes (CCs) of healthcare- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) in the USA. Most isolates (40/66) were identified as CC 5, while remaining isolates represented CCs 1, 8, 30, 45, 59, 133, and five isolates were not S. aureus.

comK prophage junction fragments as markers for Listeria monocytogenes genotypes unique to individual meat and poultry processing plants and a model for rapid niche-specific adaptation, biofilm formation, and persistence.

Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking the comK prophage (P < 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated that the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defective comK prophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as "adaptons," as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explain Listeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety.

Detecting the form of selection in the outer membrane protein C of Enterobacter aerogenes strains and Salmonella species.

The types of selective pressure operating on the outer membrane protein C (ompC) of Enterobacter aerogenes strains, the causative agent for nosocomial infections, and Salmonella sp., the hazardous pathogen are investigated using the maximum likelihood-based codon substitution models. Although the rate of amino acid replacement to the silent substitution (omega) across the entire codon sites of ompC of E. aerogenes (omega=0.3194) and Salmonella sp. (omega=0.2047) indicate that the gene is subjected to purifying selection (i.e. omega<1), approximately 3.7% of ompC codon sites in E. aerogenes (omega=21.52) are under the influence of positive Darwinian selection (i.e. omega>1). Such contrast in the intensity of selective pressures in both pathogens could be associated with the differential response to the adverse environmental changes. In E. aerogenes, majority of the positively selected sites are located in the hypervariable cell-surface-exposed domains whereas the trans-membrane domains are functionally highly constrained.

Dynamic evolutionary pattern of alpha2-macroglobulin in a model organism, the zebrafish (Danio rerio).

Studies of alpha-2-macroglobulin (alpha(2)M), a universal protease inhibitor, have indicated that it plays a unique and critical role in the innate immune system of vertebrate and invertebrate animals. The distinctive mechanism of pathogen inhibition--through physical entrapment of the pathogen-derived protease--makes alpha(2)M an ideal candidate for molecular evolutionary analysis. Furthermore, recent studies revealed that the Osteichthyes are characterized by levels of alpha(2)M diversification that exceed those recorded in other animal groups. Our study of Danio rerio (zebrafish) indicated that (1) two distinct lineages of alpha(2)M and alpha(2)M-like isoforms exist and (2) at least some codons in the functional domains of alpha(2)M have been subjected to positive Darwinian selection. The findings of several hot-spots for nonsynonymous substitutions in the two functional domains such as bait region and receptor binding domain, suggest that host-immune selection have played a dominant role in these two genomic regions of alpha(2)M. The presence of two, non-monophyletic alpha(2)M lineages in zebrafish provides compelling evidence of an ancient gene duplication event. The accelerated rate of nucleotide substitution in the functional domains of alpha(2)M is consistent with similar observations of other immune system components.

Molecular diversity and evolution of myticin-C antimicrobial peptide variants in the Mediterranean mussel, Mytilus galloprovincialis.

Mussels have diverse groups of cysteine rich, cationic antimicrobial peptides (AMPs) (defensins, mytilins, myticins, and mytimycin) that constitute an important component of their innate immune defence. Despite the identification and characterization of these AMPs in mussels, the underlying genetic mechanisms that maintain high diversity among multiple variants of the myticin-C isoform are poorly understood. Using phylogeny-based models of sequence evolution and several site-by-site frequency spectrum statistical tests for neutrality, herein we report that positive selection has been the major driving force in maintaining high diversity among the allelic-variants of the myticin-C AMP of Mytilus galloprovincialis. The statistical tests rejected the hypothesis that all polymorphism within myticin-C loci is neutral. Although a majority of the codons constrained to purifying selection (rate of amino acid replacement to the silent substitution, omega < 1), approximately 8% of the codons with omega approximately equal to 5.5 are under positive selection (omega > 1), thus indicating adaptive evolution of certain amino acids. Direct interaction of these peptides with the surrounding pathogens and/or altered/new pathogens in the changing environment is the likely cause of molecular adaptation of certain amino acid sites in myticin-C variants.

Detecting molecular adaptation at individual codons in the pattern recognition protein, lipopolysaccharide- and beta-1,3-glucan-binding protein of decapods.

Pattern recognition proteins play an important role in the innate immune response of invertebrates. Herein we report the evolutionary relationships among Gram-negative bacteria binding proteins (GNBPs) that were previously identified and characterized from a wide array of invertebrates. Our results, together with those obtained in previous studies, indicate that decapod lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP/BGBP) has retained the crucial components for glucanase activity, and shares a common ancestor with GNBPs, as well as with the glucanase proteins of a wide range of invertebrates, rather than with GNBPs of some arthropods. However, experimental evidence of earlier studies suggested a lack of glucanase activity by these proteins, thus implying that during evolutionary time these proteins might have lost their glucan binding protein, but retained their glucan binding activity. The present results have also revealed that although a vast majority of the decapod LGBP/BGBP codons are constrained to purifying selection, certain codons are shown to have a higher rate of nonsynonymous substitutions per nonsynonymous site (dN) than synonymous substitutions per synonymous site (dS), indicating these codons have evolved adaptively (dN/dS>1). Although purifying selection (dN/dS<1) appears to be the major driving force in the evolution of a vast majority of LGBP/BGBP codons in decapods, the findings of several hotspots for nonsynonymous substitutions in this protein indicate host immune selection might play an important role in maintaining diversity among these ecologically diversified decapod species.

Detecting molecular adaptation at individual codons in the glycoprotein gene of the geographically diversified infectious hematopoietic necrosis virus, a fish rhabdovirus.

Salmonid fishes, the principal hosts of the infectious hematopoietic necrosis virus (IHNV), are a candidate species for aquaculture in many countries. IHNV causes an acute disease resulting in severe economic loss in salmonid fish farming. Previous phylogenetic analyses revealed the existence of multiple genogroups of this virus throughout the geographical range of its host. Here, we report the importance of natural selection in shaping the evolution of certain codons at the surface glycoprotein (G-protein) gene of this virus. Maximum likelihood (ML)-based codon substitution analyses revealed that approximately 2.8% of the codons for the entire G-protein are shown to have higher nonsynonymous substitution per nonsynonymous site (dn) than the synonymous substitutions per synonymous site (ds) (dn/ds=omega>4.335). Thus, the data suggest that positive selection (omega>1) is the major driving force in the evolution of certain codons. However, majority of these positively selected sites cannot be mapped to the regions of antigenic determinants of IHNV. Based on the reports of previous studies, epitopes with positively selected sites are immunodominant and viruses can escape from immune responses by producing antigenic variation at positively selected sites, therefore, vaccines directed against these neutralizing epitopes of IHNV that consist of no positively selected sites will be more effective. Some of the positively selected sites showed radical change in amino acids with respect to their charge and polarity; however, it is unclear how these changes affect the fitness of the virus.

Positive natural selection in the evolution of human metapneumovirus attachment glycoprotein.

Human metapneumovirus (hMPV), a newly discovered virus of the family Paramyxoviridae, has been associated with upper and lower respiratory tract infections in different age groups in many countries. The putative attachment (G) glycoprotein of this virus was previously reported to have shown more extensive nucleotide and deduced amino acid sequence polymorphism than any other genomic regions of this virus, leading to four sub-lineages. Using a maximum likelihood-based codon substitution model of sequence evolution, here we report that sequences of extracellular domain of 8 amino acid sites in lineage 1a, and 3 amino acid sites each in lineage 1b, 2a, and 2b have a higher rate of nonsynonymous substitutions (d(N)) than the synonymous substitutions (d(S)) with a posterior probability above 0.95, thus suggesting the evidence of adaptive evolution driven by Darwinian selection. Although it is unclear whether these amino acid adaptations are driven by differential immune pressure or some other factors, identification of these positively selected amino acid sites would help in better screening using epitope mapping technology to identify and localize the sites that can be recognized by the immune system. We also observed surprisingly higher nucleotide substitution rates per site, per year for each lineage of hMPV than the rates that were previously reported for the human respiratory syncytial virus, suggesting rapid evolutionary dynamics of hMPV.

Evidence for positive Darwinian selection on the hepcidin gene of Perciform and Pleuronectiform fishes.

Hepcidin is a small cysteine-rich peptide that plays an important role in antimicrobial activity and in maintaining iron homeostasis in vertebrates. Here we report on the underlying mechanism that maintains high sequence diversities among the hepcidin-like variants of perciform and pleuronectiform fishes. In contrast to mammals, maximum likelihood-based codon substitution analyses revealed that positive Darwinian selection (nonsynonymous to synonymous substitution, omega > 1) is the likely cause of accelerated rate of amino acid substitutions in the hepcidin mature peptide region of these fishes. Comparison of models incorporating positive selection (omega > 1) at certain sites with models not incorporating positive selection (omega < 1) failed to reject (p = 0) the evidence of positive selection among the codon sites of percifom and pleuronectiform hepcidin. The adaptive evolution of this peptide in perciform and pleuronectiform fishes might be directed by pathogens when the host is exposed to new habitats/environments.

Effect of environmental parameters on immune response of the Indian spiny lobster, Panulirus homarus (Linnaeus, 1758).

The high export value of the Indian spiny lobster Panulirus homarus increasingly attracts the aquaculturists for farming and fattening. However, lack of knowledge on the effect of environmental parameters on the immune system of this animal could result in high mortality, which ultimately may cause major loss to the industry. Here, we report the effect of salinity (20, 25, 35, 40, and 45 per thousand), pH (5.0, 8.0, and 9.5), dissolved oxygen (DO) (1 and 5 mg L(-1)), and ammonia-N concentration (0, 0.5, 1.5 and 3 mg L(-1)) on the immune response of P. homarus measured in the haemolymph in terms of Total Haemocyte Count (THC), phenoloxidase (PO) activity, and NBT-reduction. Our data showed significant reduction (P<0.05) in THC, and NBT-reduction at lower (20 per thousand) and higher (45 per thousand) salinities. However, PO activity showed significant disparity, showing an increasing trend from 20 to 45 per thousand. Significant reduction (P<0.05) in THC and PO activity under acidic and alkaline conditions, under hypoxic condition (1 mg L(-1)), and at the higher ammonia-N concentrations than their respective optimal conditions were observed. Thus, suggesting that extreme environmental parameters can induce modifications in the immune system of the spiny lobster P. homarus, which may enhance their susceptibility to opportunistic pathogens. The humoral parameters such as THC, PO activity, and NBT-reduction can be used as potential stress indicators for healthy management of spiny lobsters.

Adaptive evolution after duplication of penaeidin antimicrobial peptides.

Penaeidin antimicrobial peptides in penaeid shrimps are an important component of their innate immune system that provides immunity against infection caused by several gram-positive bacteria and filamentous fungal species. Despite the knowledge on the identification and characterization of these peptides in penaeid shrimps, little is known about the evolutionary pattern of these peptides and the underlying genetic mechanisms that maintain high sequence diversities in the penaeidin gene family. Based on the phylogenetic analyses and maximum likelihood-based codon substitution analyses, here we present the convincing evidence that multiple copies of penaeidins have evolved by gene duplication, and positive Darwinian selection (adaptive evolution) is the likely cause of accelerated rate of amino acid substitutions among these duplicated genes. While the average ratio of non-synonymous to synonymous substitutions (omega) for the entire coding region of both active domains is 0.9805, few codon sites showed significantly higher omega (3.73). The likelihood ratio tests that compare models incorporating positive selection (omega>1) at certain codon sites with models not incorporating positive selection (omega<1), failed to reject (p=0) the evidence of positive Darwinian selection. The rapid adaptive evolution of this gene family might be directed by the pathogens and the faster rate of amino acid substitutions in the N-terminal proline-rich and C-terminal cysteine-rich domains could be due to their direct involvement in the protection against pathogens. When the host expose to different habitats/environment an accelerated rate of amino acid substitutions in both the active domains may also be expected.

Pattern of nucleotide substitution and divergence of prophenoloxidase in decapods.

Despite the unprecedented development in identification and characterization of prophenoloxidase (proPO) in commercially important decapods, little is known about the evolutionary relationship, rate of amino acid replacement and differential selection pressures operating on proPO of different species of decapods. Here we report the evolutionary relationship among these nine decapod species based on proPO gene and types of selective pressures operating on proPO codon sites. Our analyses revealed that all the nine decapod species shared a common ancestor. The mean percentage sequence divergence at proPO gene was 34.4+/-0.6%. Pairwise estimates of nonsynonymous to synonymous ratio (omega) for Homarus americanus-H. gammarus is greater than one, therefore indicating adaptive evolution (functional diversification) of proPO in these two species. In contrast, strong purifying selection (omega<1) was observed in all other species pairs. However, phylogenetically closely related decapods revealed relatively higher omega value (omega=0.15+/-0.3) than the distantly related species pairs (omega=0.0075+/-0.005). These discrepancies could be due to higher fixation probability of beneficial mutation in closely related species. Maximum likelihood-based codon substitution analyses revealed a strong purifying selection operating on most of the codon sites, therefore suggesting proPO is functionally constrained (purifying selection). Codon substitution analyses have also revealed the evidence of strong purifying selection in haemocyanin subunits of decapods.

Positive Darwinian selection on crustacean hyperglycemic hormone (CHH) of the green shore crab, Carcinus maenas.

The tissue-specific expression and differential function of the crustacean hyperglycemic hormone (CHH) in Carcinus maenas indicate an interesting evolutionary history. Previous studies have shown that CHH from the sinus gland X-organ (XO-type) has hyperglycemic activity, whereas the CHH from the pericardial organ (PO-type) neither shows hyperglycemic activity nor it inhibits Y-organ ecdysteroid synthesis. Here we examined the types of selective pressures operating on the variants of CHH in Carcinus maenas. Maximum likelihood-based codon substitution analyses revealed that the variants of this neuropeptide in C. maenas have been subjected to positive Darwinian selection indicating adaptive evolution and functional divergence among the CHH variants leading to two unique groups (PO and XO-type). Although the average ratio of nonsynonymous to synonymous substitution (omega) for the entire coding region is 0.5096, few codon sites showed significantly higher omega (10.95). Comparison of models that incorporate positive selection (omega > 1) with models not incorporating positive selection (omega <1) at certain codon sites failed to reject (p=0) evidence of positive Darwinian selection.