**Short Biography of Author**

**Ioan Balint.** Dr. Ioan Balint is a Senior Researcher and currently, the Director of the "Ilie Murgulescu" Institute of Physical-Chemistry of the Romanian Academy. Dr. Balint obtained his PhD in Chemistry in 1996 at the same institute. He obtained three postdoctoral stays, one of them at the Pierre et Marie Marie Curie University in France (1998–1999), and another two at the Department of Environmental Chemistry and Catalysis in the group of Prof. Ken-ichi Aika, of the Tokyo Institute of Technology in Japan (2000–2005). He was several times, an invited professor at Tokyo Institute of Technology and Tokyo Technical University, in Japan and also gained an UNESCO Scholarship at the same institute. Dr. Balint has an extensive research background encompassing various topics such as surface science: non-isothermal gas desorption kinetic from supported-metal catalysts; solid defect chemistry; water–gas shift reaction on surface of simple and doped ionic oxides; material synthesis: mesoporous nano oxides; mono/bimetallic metal nanoparticles; catalysis: catalytic combustion of hydrocarbons; oxidative coupling of methane; hydrogenation and oxidative conversion of conversion hydrocarbons; structure-sensitive reactions; nitrate and nitrite abatement; photocatalysis/light harvesting: water and air depollution; water splitting; light-induced reactive oxygen species generation; solar cell. He was project director for 5 national grants, and has

been as expert member in many national/international committees. Dr. Balint has published over 100 peer-reviewed papers in well-recognized scientific journals, and has an H-index of 21.

**Monica Pavel.** Dr. Monica Pavel is a senior researcher at the "Ilie Murgulescu" Institute of Physical-Chemistry of the Romanian Academy. She obtained a BSc degree in chemistry and physics in 2005 at the University of Pitesti, Romania, and a MSc degree (2006) in materials science, nanomaterials and multimaterials from the National Polytechnic Institute of Toulouse, France. From October 2007 to November 2010, she carried out her PhD studies in materials chemistry at the University of Lyon 1—Institute of Researches for Catalysis and Environment of Lyon in France under the supervision of Dr. Pavel Afanasiev. She continued as a postdoctoral researcher (2011–2013) at the University of Bucharest, Romania, with a short internship at the Institute Charles Gerhardt Montpellier, France. Her research focuses on the controllable synthesis of transition-mixed oxides and 2D materials by various methods (e.g., molten salts, organic solvents, co-precipitation, solid state reactions, ion-exchange reactions); the photocatalytic removal of pollutants (chlorinated/organic compounds, antibiotics, etc.); combustion of light alkanes, and the total oxidation of volatile organic compounds (VOCs).

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**6. Conclusions**

GMM clustering with MCLUST identified three Gaussian components of *Swift*/BAT and *Fermi*/GBM bursts in the duration-hardness plane. The third component resembled the intermediate duration group identified in previous studies. However, combining components, based on an entropy criterion, identified a short and long duration class only for both samples.

This study highlights the drawbacks of fitting GRB populations with model-based methods. Similar model-based fitting methods, including the log-normal fit procedures applied to GRB duration distributions, have exhibited components thought to be identified incorrectly due to the inherently skewed distribution of long GRB durations [31,36]. Table 1 highlights the diversity of results from model-based studies.

The lack of consensus regarding a definitive number of GRB classes, both in the analysis presented here and in previous studies of GRB catalogues, is a motivator for a modelindependent analysis of GRB light curves. The light curves may also contain more information than the summary data provided by the GRB catalogues. Fourier analysis of the *Swift*/BAT GRB light curves by Jespersen et al. [101] identified two classes of bursts. Following on from the analysis presented in this paper is a wavelet-based feature extraction analysis of GRB light curves from *Swift*/BAT, BATSE and *Fermi*/GBM (Salmon et al. [102], in preparation).

**Author Contributions:** Conceptualization, L.S., L.H. and A.M.-C.; methodology, L.S., L.H. and A.M.-C.; software, L.S.; validation, L.S., L.H. and A.M.-C.; formal analysis, L.S.; investigation, L.S.; resources, L.S., L.H. and A.M.-C.; data curation, L.S.; writing—original draft preparation, L.S.; writing—review and editing, L.H. and A.M.-C.; visualization, L.S.; supervision, L.H. and A.M.-C. ; project administration, L.H. and A.M.-C.; funding acquisition, L.H. and A.M.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** L.S. acknowledges the Irish Research Council Postgraduate Scholarship No GOIPG/2017/1525. L.H. acknowledges support from Science Foundation Ireland (Grant number 19/FFP/6777) and the EU H2020 (Grant agreement 871158).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** This paper makes use of the *Swift*/BAT catalogue [76], which can be accessed through the online *Swift*/BAT Gamma-ray Burst Catalogue 1 . The *Fermi*/GBM catalogue is accessed through the *Fermi*/GBM data tools [40].

**Acknowledgments:** This research made use of the following Python packages: NumPy [103], Matplotlib [104] and pandas [105,106]. The authors are grateful to Brendan Murphy and Michael Fop for their assistance with MCLUST.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

## **Note**

1 https://swift.gsfc.nasa.gov/results/batgrbcat/, (accessed on 16 June 2022).


**Author details**

Mateus Pereira Gonzatto1 \* and Júlia Scherer Santos2

1 Department of Agronomy, Federal University of Viçosa, Viçosa, Brazil

2 Faculty of Pharmacy, Department of Pharmaceutical Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Introductory Chapter: World Citrus Production and Research DOI: http://dx.doi.org/10.5772/intechopen.110519*


*Edited by Oscar Grillo and Gianfranco Venora*

Driven by the increasing necessity to define the biological diversity frame of widespread, endemic and threatened species, as well as by the stimulating chance to describe new species, the study of the evolutive and spatial dynamics is in constant execution. Systematic overviews, biogeographic and phylogenic backgrounds, species composition and distribution in restricted areas are focal topics of the 15 interesting independent chapters collected in this book, chosen to offer to the reader an overall view of the present condition in which our planet is.

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The Dynamical Processes of Biodiversity -

Case Studies of Evolution and Spatial Distribution

The Dynamical Processes of

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*Edited by Oscar Grillo and Gianfranco Venora*


**5.3 Other findings**

Despite major differences between herpesvirus genomes, all the herpesviruses studied thus far have a similar number of essential genes, but varying number of nonessential genes. For example, our study suggests that the VZV genome encodes 44 essential genes and 26 nonessential genes. The herpes simplex virus 1 (HSV-1) genome encodes 37 essential genes and 48 nonessential genes (Roizman et al., 2007). Similarly, the human cytomegalovirus (HCMV), one of the largest human DNA viruses, has a genome that encodes 45 essential genes and 118 nonessential genes (Dunn et al, 2003). Furthermore, 26 of the 44 essential VZV genes have essential homologues in HSV and 18 of the 44 have essential gene homologues in HCMV, alluding that some essential genes may perform core functions for all of these herpesviruses.

Various studies, cumulatively, have found that the essential VZV ORFs encode genes for viral structural proteins, transcriptional regulatory proteins, and enzymes involved in DNA replication. The majority of these crucial ORFs encode proteins with imperative functions in maintaining the viral life cycle. For example, some ORFs are a part of the viral tegument and encode immediate-early proteins with transcriptional regulatory activity (Perera et al., 1992; Defechereux et al., 1993; Moriuchi et al., 1994). Other ORFs encode phosphoproteins primarily contained in the nuclei of infected cells (Moriuchi et al., 1993). It has also been reported that most of the VZV ORFs encoding glycoproteins also belong in this group of genes indispensable for viral replication (Mallory et al., 1998;

Upon further analysis, we found that essential VZV genes have significantly different enrichment for functional categories than nonessential genes. As depicted by the distribution of functional annotations (Fig. 9A), essential VZV genes are significantly enriched for DNA replication and DNA packaging. These include genes encoding the subunits of VZV DNA polymerases, DNA binding proteins, DNA packaging proteins, and

As previously mentioned, 26 of the 70 unique VZV ORFs were deemed nonessential for VZV replication in MeWo cells (Zhang et al., 2010). Of these, 8 ORFs appeared to significantly affect viral growth. In viral growth assays, the peak signals from their corresponding plaques were at least 5-fold less than the peak signals from the WT parental strain. Furthermore, atypical morphology of virally infected cells, such as reduced plaque sizes and altered syncytia formation, were also frequently observed. Studies have shown that some of these ORFs affecting optimal growth encode the small and large subunit of ribonucleotide reductase (Heineman & Cohen, 1994) and specific phosphoproteins that are

The plaques corresponding to the remaining 18 nonessential VZV ORF deletions exhibited wild-type growth in cultured MeWo cells (Zhang et al., 2010). *In vitro* growth curve analysis for viral replication showed that these ORF deletion mutants have the same growth kinetics as their wild-type parental strain, VZVLuc. Nonessential genes, in general, are significantly

Despite major differences between herpesvirus genomes, all the herpesviruses studied thus far have a similar number of essential genes, but varying number of nonessential genes. For example, our study suggests that the VZV genome encodes 44 essential genes and 26 nonessential genes. The herpes simplex virus 1 (HSV-1) genome encodes 37 essential genes and 48 nonessential genes (Roizman et al., 2007). Similarly, the human cytomegalovirus (HCMV), one of the largest human DNA viruses, has a genome that encodes 45 essential genes and 118 nonessential genes (Dunn et al, 2003). Furthermore, 26 of the 44 essential VZV genes have essential homologues in HSV and 18 of the 44 have essential gene homologues in HCMV, alluding that some essential genes may perform

post-translationally modified by protein kinases (Reddy et al., 1998).

enriched for other and unknown functional categories (Fig. 9B).

core functions for all of these herpesviruses.

Yamagishi et al., 2008).

nucleocapsid proteins.

**5.3 Other findings**

**5.2 Nonessential VZV ORFs**

Another observation worth noting is the size of the essential ORFs as compared to the size of the nonessential ORFs. Essential ORFs are significantly larger in size, averaging 1250-bp, while nonessential ORFs have an average size of 970-bp. Moreover, the ten largest VZV ORFs are all essential, while of the 11 smallest VZV ORFs, eight are nonessential.

Fig. 9. Distribution of functional annotations for essential and nonessential genes. (A) Distribution of functional annotations for essential genes. Essential genes are significantly enriched for DNA replication (Bonferroni corrected p-value <10-4) and DNA packing (corrected p-value <10-4) functional categories. (B) Distribution of functional annotations for nonessential genes. Nonessential genes are significantly enriched for other (corrected pvalue <10-3) and unknown (corrected p-value <0.01) functional categories. Statistical significance was determined by a hypergeometric test.


*Article* **Multidrug-Resistant Methicillin-Resistant Coagulase-Negative Staphylococci in Healthy Poultry Slaughtered for Human Consumption**

**Vanessa Silva 1,2,3,4 , Manuela Caniça 5,6 , Eugénia Ferreira 5,6, Madalena Vieira-Pinto 7 , Cândido Saraiva 7 , José Eduardo Pereira 1,7,8, José Luis Capelo 9,10 , Gilberto Igrejas 2,3,4 and Patrícia Poeta 1,4,7,8,\***

**Abstract:** Coagulase-negative staphylococci are commensals that are known to be prevalent in most environments, and they are also an important reservoir of antimicrobial-resistant genes. Staphylococcal infections in animal husbandry are a high economic burden. Thus, we aimed to determine the prevalence and species diversity of methicillin-resistant coagulase-negative staphylococci (MRCoNS) in poultry slaughtered for human consumption and to study the antimicrobial resistance of the isolates. Swab samples were recovered from 220 commercial chickens, homebred chickens and quails. Species identification was performed using MALDI-TOF. Antimicrobial susceptibility testing was performed by the disc diffusion method against 14 antimicrobials. The presence of antimicrobialresistant genes was investigated by polymerase chain reaction. Totals of 11 (19.6%), 13 (20.3%), and 51 (51%) MRCoNS were isolated from commercial chickens, homebred chickens and quails, respectively. *S. lentus* was isolated from all homebred chickens, whereas 11 *S. lentus* and 2 *S. urealyticus* were isolated from commercial chickens. As for quails, the most prevalent MRCoNS were *S. urealyticus*. Almost all isolates had a multidrug-resistant profile and carried the *mecA* gene. Most isolates showed resistance to erythromycin, clindamycin, penicillin, tetracycline, ciprofloxacin and fusidic acid and harbored the *ermA*, *ermB*, *ermC*, *mphC tetK*, *tetL*, *tetM* and *tetO* genes. This study showed a frequent occurrence of multidrug resistance in MRCoNS isolated from healthy poultry in Portugal.

**Keywords:** coagulase-negative *Staphylococcus*; CoNS; antimicrobial resistance; poultry; quails; broilers

**Citation:** Silva, V.; Caniça, M.; Ferreira, E.; Vieira-Pinto, M.; Saraiva, C.; Pereira, J.E.; Capelo, J.L.; Igrejas, G.; Poeta, P. Multidrug-Resistant Methicillin-Resistant Coagulase-Negative Staphylococci in Healthy Poultry Slaughtered for Human Consumption. *Antibiotics* **2022**, *11*, 365. https://doi.org/ 10.3390/antibiotics11030365

Academic Editors: Clair L. Firth and Marc Maresca

Received: 18 January 2022 Accepted: 3 March 2022 Published: 9 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Staphylococci colonize the skin and mucous membranes of humans and are considered commensals or opportunistic pathogens [1]. By 2018, 45 species and 24 subspecies of *Staphylococcus* had been described [2]. Staphylococci are divided into two groups, coagulasepositive (CoPS) and coagulase-negative staphylococci (CoNS), according to their ability to coagulate plasma. CoPS are pathogenic species which have the coagulase enzyme that converts plasma fibrinogen into fibrin [3]. CoNS lack this enzyme and were considered, until recently, to be minor pathogens or apathogenic [4]. CoNS possess fewer virulence factors that participate in the pathogenesis of infection when compared to CoPS, such as *S. aureus*, but, in the last few decades, CoNS have emerged as common causes of nosocomial infections [4]. Within the CoNS species, *S. epidermidis*, *S. haemolyticus* and *S. saprophyticus* are examples of the most significant types of CoNS in human infections [5]. As opportunistic pathogens, CoNS generally cause infection in colonized immunocompromised individuals, patients with catheters and prosthetic implants, dialysis and oncologic patients and neonates [6]. CoNS are responsible for a broad spectrum of infections, such as invasive endocarditis, bacteremia and bone infections [6,7]. In addition, increasing rates of antibiotic resistance have been detected in CoNS, in some cases even greater than for *S. aureus*, which limits the therapeutic options available [5]. Methicillin resistance in CoNS is usually due to the expression of the *mecA* gene, which encodes an alternative binding protein 2a (PBP2a) that has a low affinity for β-lactam antibiotics, although some studies have reported the presence the *mecC* gene, a homologue of *mecA* [8–10]. The *mec* genes are located on a mobile genetic element called the Staphylococcal Cassette Chromosome *mec* (SCC*mec*). SCC*mec* elements are more diverse in methicillin-resistant CoNS when compared to *S. aureus,* and many SCC*mec* elements could not be typed using multiplex PCR [10]. Tetracycline resistance is also frequently detected in different CoNS species [11].

CoNS also colonize and infect other mammals besides humans, with *S. chromogenes*, *S. simulans* and *S. xylosus* being the principal cause of infection [11]. CoNS are frequently responsible for arthritis, cow mastitis and, less often, systemic infections in animals [12]. The presence of CoNS has been reported in pets, livestock and wild animals [13–15]. It has been shown that food of animal origin can carry CoNS and other foodborne pathogens and, besides being able to cause infection, CoNS can also cause food poisoning [16]. Both CoPS and CoNS have been associated with avian pathologies such as arthritis, osteomyelitis, pododermatitis, septicemia and blepharitis [17,18]. Nevertheless, the presence of CoPS and CoNS has also been observed in healthy poultry and poultry meat, which may act as reservoirs and vehicles of zoonotic pathogens and antimicrobial resistance [16,19]. The spread of antimicrobial resistance among commensal CoNS in healthy poultry may represent a hazard for human and animal health [11]. Studies reporting the monitorization of antimicrobial-resistant pathogens in poultry and poultry meat have been published, but most studies focus only on *S. aureus* species [20–24]. The prevalence of antimicrobialresistant pathogens in poultry, particularly staphylococci, may be due to their high consumption of antimicrobials. According to the ESVAC report, in Portugal the populationweighted mean consumption (expressed in milligrams per kilogram of estimated biomass) of antimicrobials was 175.8 mg/Kg in food-producing animals in 2020 [25]. In Portugal, the biomass-corrected consumption of third- and fourth-generation cephalosporins, quinolones, penicillin, macrolides and tetracyclines in food-producing animals was around 0.4, 7.3, 38.9, 20 and 60.4 mg/Kg [25]. Furthermore, all these antimicrobial classes were used in poultry production. Therefore, we aimed to investigate the presence of methicillin-resistant CoNS (MRCoNS) in healthy poultry for human consumption as well as the antimicrobial-resistant phenotypes and genotypes of the isolates.

#### **2. Results**

In this study, the presence of methicillin-resistant CoNS (MRCoNS) was detected in 71 (32.3%) of the 220 birds tested (Table 1). The co-carriage of two different species was identified in four animals, and 67 birds carried only one staphylococcal species. Cocarriage of MRCoNS species was identified only among quail samples, and the pattern of co-carriage was as follows: *Staphylococcus sciuri*/*S. urealyticus* (*n* = 2), *Staphylococcus lentus*/*S. urealyticus* and *Staphylococcus lentus*/*Staphylococcus haemolyticus*. A total of 75 MRCoNS were recovered and identified as *S. lentus* (*n* = 26), *S. urealyticus* (*n* = 21), *S. sciuri* (*n* = 15) and *S. haemolyticus* (*n* = 3). *S. haemolyticus* was exclusively isolated from quails. Chickens, both commercial and homebred, were mainly colonized by *S. lentus,* while *S. urealyticus* was the most frequently detected species in quails, followed by *S. lentus*. Quails were colonized significantly more frequently by MRCoNS than homebred chickens. Furthermore, the prevalence of *S. lentus* and *S. urealyticus* was significantly higher than that of *S. haemolyticus*. Results of the prevalence of each staphylococcal species are shown in Supplementary Figure S1.

**Table 1.** Number of animals sampled, frequency and diversity of CoNS species detected among healthy poultry.

Table 2 shows the antimicrobial-resistant phenotypes and genotypes of MRCoNS, while the detailed characterization of each isolate is summarized in Supplementary Table S1. The percentage of resistance to each antibiotic is shown in Figure 1. All isolates showed phenotypic and genotypic resistance to antibiotics, with 73 (97.3%) isolates displaying a multidrug-resistant profile since they showed resistance to at least three different classes of antimicrobials. The multidrug-resistance pattern was as follows: 15 (20%) isolates were resistant to 3 classes, 27 (26%) to 4 classes, 17 (22.7%) to 5 classes, 12 (16%) to 6 classes and 2 (2.7%) to 7 classes of antimicrobials. The non-multiresistant isolates were both *S. lentus* and were isolated from chickens. Both isolates showing resistance to seven antimicrobial classes were isolated from quails. The *mec*A gene was detected in all isolates, including those that were susceptible to cefoxitin. Totals of 11 *S. lentus*, 21 *S. urealyticus*, 14 *S. sciuri* and 3 *S. haemolyticus* were phenotypically resistant to penicillin, but the mechanism of penicillin resistance could not be identified. Resistance to aminoglycosides was detected in 40% of the isolates and was mediated by the *aph*(30 )-IIIa, *ant*(40 )-Ia and *str* genes in different combinations. All *S. lentus* and *S. urealyticus* were resistant to macrolides and lincosamides, while 14 *S. sciuri* and 2 *S. haemolyticus* showed resistance to this antimicrobial class. Macrolide-lincosamide resistant isolates harbored the *ermA*, *ermB*, *ermC* and *mphC* genes alone or in different combinations: *ermB* (*n* = 5); *ermC* (*n* = 11); *mphC* (*n* = 3); *ermC* and *mph*C (*n* = 27); *ermA*, *ermC* and *mphC* (*n* = 6); *ermB*, *ermC* and *mphC* (*n* = 10); *erm*B and *mphC* (*n* = 8); *ermA* and *ermC* (*n* = 1); *ermA*, *ermB*, *ermC* and *mphC* (*n* = 1); and *ermA*, *ermB* and *mphC* (*n* = 1). Tetracycline resistance, which was detected in all *S. urealyticus*, *S. sciuri* and *S. haemolyticus*, and in 25 (69.4%) *S. lentus*, was mediated by the tet*K*, tet*L*, tet*M* and/or *tetO* genes. The *tetL* gene was the most frequent, followed by the *tetK*. The *cat*p194 encoding resistance to chloramphenicol was detected in one *S. lentus* isolate. Resistance to trimethoprim-sulfamethoxazole was detected in 10 isolates. Some *S. lentus* isolates harbored a combination of *dfrK* and *dfrD* genes, while *S. sciuri* and *S. haemolyticus* carried only the *dfrK*. One *S. sciuri* exhibited resistance to linezolid, mediated by the *cfr* gene. None of the isolates showed resistance to vancomycin.

**Table 2.** Antimicrobial-resistant genes identified among the CoNS isolated from poultry.

Abbreviations. C: chloramphenicol; CD: clindamycin; CIP: ciprofloxacin; ERY: erythromycin; FD, fusidic acid; FOX: cefoxitin; PEN: penicillin; SXT: trimethoprim-sulfamethoxazole; TET: tetracycline; CN: gentamicin; KAN: kanamycin; TOB: tobramycin; LNZ: linezolid. Note: the superscript number after each antibiotic and gene indicates the number of strains showing resistance to that antibiotic and harboring that gene, respectively. *dfrK*. One *S. sciuri* exhibited resistance to linezolid, mediated by the *cfr* gene. None of the isolates showed resistance to vancomycin.

**Figure 1.** Percentage of resistance to each antibiotic by MRCoNS isolated from poultry. **Figure 1.** Percentage of resistance to each antibiotic by MRCoNS isolated from poultry.

#### **Table 2.** Antimicrobial-resistant genes identified among the CoNS isolated from poultry. **3. Discussion**

**Species Number of Isolates Antimicrobial Resistance Phenotype Genotype**  *S. lentus* 36 PEN11, FOX4, CIP11, CN2, TOB14, KAN9, ERY35, CD36, TET25, C4, FD12, SXT6 *mecA36*, *ermA8*, *ermB8*, *ermC28*, *mphC29*, *aph(3′)-IIIa9*, *ant(4′)-Ia12*, *str2*, *tetL19*, *tetK14*, *tetO1*, *tetM2*, *catp1941*, *dfrK6*, *dfrD2 S. urealyticus* 21 PEN21, FOX18, CIP3, CN4, TOB6, KAN5, ERY21, CD21, TET21, C3, FD17 *mecA21*, *ermA1*, *ermB7*, *ermC19*, *mphC16*, *aph(3′)-IIIa5*, *ant(4′)-Ia2*, *str2*, *tetL17*, *tetK18*, *tetO13*, *tetM4 S. sciuri* 15 PEN14, FOX6, LNZ1, CIP3, TOB8, KAN4, ERY14, CD14, TET15, C2, *mecA15*, *cfr1*, *ermB9*, *ermC7*, *mphC9*, *aph(3′)-IIIa3*, *ant(4′)-Ia7*, *str1*, *tetL11*, *tetK12*, *tetO2*, *tetM3*, MRCoNS in livestock was first reported in healthy chickens in Japan in 1996. Despite the increasing interest in CoNS in recent years, there is very limited information on their prevalence and resistance profiles in poultry production, and information is even more limited regarding MRCoNS. In our study, we investigated the presence of MRCoNS in healthy quails and commercial and homebred chickens. Among the 220 birds tested, 71 (32.3%) carried at least one CoNS, which is in accordance with the results obtained by Marek et al. [26]. CoNS colonized 47% and 20% of the quails and chickens, respectively. This carriage frequency was higher than the one obtained by Younis et al., who found a prevalence of CoNS in quails and chickens of 8.75% and 7.14%, respectively [27]. A study conducted with turkey samples found a frequency of CoNS of 15.6%, which is also lower than the one obtained in this study [28]. Other studies found a higher frequency of CoNS in poultry [18,29]. Nevertheless, it is important to point out that in our study all samples were only screened for the presence of MRCoNS, which may have contributed to a higher frequency of CoNS. Furthermore, some studies focused only on diseased animals

FD10, SXT2

PEN3, FOX1, CIP2, TOB2, KAN1, ERY2, CD2, TET3, FD2, SXT2

Abbreviations. C: chloramphenicol; CD: clindamycin; CIP: ciprofloxacin; ERY: erythromycin; FD, fusidic acid; FOX: cefoxitin; PEN: penicillin; SXT: trimethoprim-sulfamethoxazole; TET: tetracycline; CN: gentamicin; KAN: kanamycin; TOB: tobramycin; LNZ: linezolid. Note: the superscript number after each antibiotic and gene indicates the number of strains showing resistance to that

MRCoNS in livestock was first reported in healthy chickens in Japan in 1996. Despite the increasing interest in CoNS in recent years, there is very limited information on their prevalence and resistance profiles in poultry production, and information is even more limited regarding MRCoNS. In our study, we investigated the presence of MRCoNS in

*dfrK1*

*mecA3*, *ermB1*, *ermC2*, *mphC2*, *aph(3′)-IIIa2*, *ant(4′)-Ia1*, *str1*, *tetL3*, *tetK1*, *dfrK1*

*S. haemolyticus* 3

**3. Discussion**

antibiotic and harboring that gene, respectively.

that would most likely have been discarded in the slaughterhouse and would not have reached the final consumer. In our study, only four different species of CoNS were detected: *S. lentus* (*n* = 26), *S. urealyticus* (*n* = 21), *S. sciuri* (*n* = 15) and *S. haemolyticus* (*n* = 3). The predominant CoNS species found in our study included those commonly found in skin microbiota in chickens [29,30]. The occurrence of the staphylococci species among poultry samples appears to vary widely. *Pyzik* et al. detected a high number of CoNS species in diseased broiler chickens and turkeys, with *S. cohnii* being the most frequent followed by *S. saprophyticus* and *S. epidermidis* [29]. In accordance with our results, Saha et al. found a higher occurrence of *S. lentus* in poultry samples [30]. Boamah et al. reported a frequency of 42.97% *S. sciuri*, 35.94% *S. lentus*, 4.30% *S. xylosus*, 3.91%, *S. haemolyticus* 3.91%, 1.95% *S. saprophyticus* and 0.39% *S. cohnii* [31]. A study conducted in Brazil found that most CoNS from chickens were *S. gallinarum* followed by *S. simulans* [18]. In a report by El-Nagar et al., the majority of CoNS were *S. xylosus* [32]. Marek et al. found a higher occurrence of S. epidermidis in poultry in Poland [26]. Finally, *S. hominis* followed by *S. xylosus* and *S. lentus* were the most frequently detected species in quail eggs [33]. Yet, most studies have reported the presence of *S. sciuri*, *S. lentus* and *S. cohnii*. It has been shown that some species of CoNS, such as *S. sciuri*, *S. xylosus* or *S. cohnii,* are considered important poultry pathogens, particularly when associated with antimicrobial resistance [29]. Furthermore, most of these CoNS species are considered an issue of meat safety rather than the classical poultry pathogens [29].

The most common species found among poultry in this study was *S. lentus*. This species is considered an animal pathogen and has been detected among livestock, pets, wild animals and retail meats [13,16,34,35]. Nevertheless, *S. lentus* has also been responsible for a wide range of human infections and its clinical relevance seems to be increasing [36]. *S. urealyticus* was the second most common CoNS species found in poultry and it was mostly detected in quail samples. This CoNS species has been regarded as a commensal organism and is not usually involved in severe infections [37]. *S. urealyticus* strains of animal origin were shown to have multiple phenotypic resistances and carry several antimicrobial resistance genes [38]. All CoNS isolated in this study harbored the *mecA* gene, and the methicillin resistance of the isolates was confirmed. However, most *S. lentus* and *S. sciuri* isolates were phenotypically susceptible to cefoxitin. It has been shown that the staphylococcal species belonging to the *S. sciuri* group, which include *S. sciuri*, *S. fleurettii*, *S. lentus*, *S. stepanovicii* and *S. vitulinus*, carry a close homologue to the *mecA* gene, which does not confer resistance to β-lactam antibiotics [39]. Accordantly, almost all *S. urealyticus* had phenotypic resistance to cefoxitin. Multidrug resistance was exhibited in almost all isolates, which is in accordance with other studies conducted with poultry samples [27–29]. Although the European Union banned the use of antibiotics for growth promotion in livestock in 2006, and several other measures have been taken since then, it is estimated that over 60% of all antimicrobials produced are used in livestock comprising poultry [40]. Higher resistance levels were detected among quails, including two isolates resistant to seven antimicrobial classes, which may be explained by the fact that in Portugal the legislation for antibiotics administration in quails is not as well-regulated as that for other poultry, such as chickens; thus, antibiotics may be administrated indiscriminately to quails, leading to an increase in antimicrobial resistance [20]. Only one isolate, *S. sciuri*, was resistant to linezolid and carried the *cfr* gene. This gene was first detected in a bovine *S. sciuri* [41]. Although uncommon, resistance to linezolid mediated by the *cfr* gene is worrisome, since this gene confers cross-resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A antibiotics [42,43]. Studies reporting the *cfr* gene in poultry identified it in *S. lentus*, *S. urealyticus*, *S. arlettae*. *sciuri* and *S. simulans* [39,44,45]. Furthermore, a low frequency of this gene has been reported in CoNS from poultry [39]. Resistance to macrolides and lincosamides was detected in all isolates, except for one *S. sciuri* and one *S. haemolyticus*, and it was mediated by the *ermA*, *ermB*, *ermC* and *mphC* genes. Both *ermC* and *mphC* genes were carried by 56 isolates. Phosphotransferases are encoded by the *mphC* gene which confers resistance to erythromycin and other macrolides but not

to lincosamides [46]. Nevertheless, the *erm* genes confer cross-resistance to macrolides, lincosamides and streptogramins B [46]. Although the *ermA* and *ermC* genes are the most frequent *erm* genes in staphylococci, the *ermA* gene was only detected in the *S. lentus* and *S. urealyticus* isolates, while *ermB* was identified in all MRCoNS species in this study. Other studies reported similar results for the frequency of *erm* genes in poultry [28,39]. A study by Syed et al. investigated the resistance of staphylococci in poultry intestines and reported a lower frequency of resistance to macrolides and lincosamides, but the *erm*C gene was also the most prevalent [47]. In the same study, resistance to tetracycline was detected in more than half of the isolates encoded by the *tetK* and *tetM* genes [47]. In our study, resistance to tetracycline was detected in 85.3% of the isolates, including all *S. sciutri*, *S. urealyticus* and *S. haemolyticus*, and in 25 out of 36 *S. lentus*, which was similar to the findings of other studies [28,31,48]. The high frequency of tetracycline resistance in poultry samples may be due to the fact that, according to the ECDC/EFSA/EMA report, tetracycline and penicillin were the most prescribed antibiotics for food-producing animals in 2017 [49]. Among the genes that confer resistance to tetracycline, *tet*L (*n* = 50) was the most prevalent, followed by *tetK* (*n* = 45), *tetO* (*n* = 16) and *tetM* (*n* = 9). Similar results were obtained by Lee et al. in a study that investigated the *tet* genes in poultry meat [16]. In contrast, in a study by Nemeghaire et al. *tet*M was the most common gene among *S. sciuri* from healthy chickens [39]. However, due to the lack of studies investigating the prevalence of resistant genes in CoNS from poultry, it is difficult to make a direct comparison. Fusidic acid was detected in 54.6% of the isolates but none of the resistance genes tested were found, which suggests the presence of other resistant genes. Indeed, in a study by Chen et al. none of the fusidic acid-resistant *S. urealyticus* possessed *fusB*, *fusC* or *fusD* genes; instead, *S. urealyticus* isolates carried the novel *fusF* gene, which seems to be an intrinsic factor in *S. urealyticus* and may not be conserved in another subspecies [50]. Resistance to vancomycin was not detected in this study, which was unsurprising since vancomycin-resistant staphylococci are rare and, as far as we know, in Portugal there is only one study reporting a vancomycin intermediate-resistant *S. aureus* isolated from a human infection [51].

In general, penicillin and tetracycline are extensively used for the treatment of staphylococcal infections in poultry [52]. In our study, we also found higher levels of resistance to those antimicrobial agents. The ingestion of poultry meat contaminated with staphylococci may lead to food poisoning. Furthermore, the handling or ingesting of staphylococci contaminated meat is a potential risk factor for colonization by methicillin-resistant staphylococci [53]. Our findings show that the frequency of multidrug-resistant staphylococci in poultry is alarming and may represent a public health problem.

### **4. Materials and Methods**

#### *4.1. Sample Collection and Bacterial Isolates*

During the month of February 2020, a total of 220 samples were collected from poultry in a Portuguese slaughterhouse. Swab samples were collected from the cloaca and trachea of 100 quails, 50 commercial chickens and 70 homebred chickens. Batches of quails, homebred and commercial chickens arrived at the slaughterhouse 3 days a week and around 36,000 quails, 3500 homebred and 8000 commercial chickens were slaughtered each day. Four samples were recovered from each batch. The swabs were inserted into tubes containing brain heart infusion (BHI) broth with 6.5% of NaCl and incubated at 37 ◦C under aerobic conditions for 24 h. The inoculum was then seeded onto ORSAB agar plates supplemented with 2 mg/mL of oxacillin, incubated at 37 ◦C and examined after 24 h to 48 h. Up to three colonies per plate with different colors and morphology were recovered and further investigated. The staphylococci species identification was performed by matrix-assisted laser desorption/ionization time-of-flight coupled to time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) as described by Dubois et al. [54].

#### *4.2. Phenotypic Antibiotic Resistance Testing*

Antibiotic susceptibility profiles were determined for all of isolates by the Kirby–Bauer disc diffusion method on Mueller Hinton agar. The tested antibiotics included: cefoxitin (30 µg), chloramphenicol 132 (30 µg), ciprofloxacin (5 µg), clindamycin (2 µg), erythromycin (15 µg), fusidic acid (10 133 µg), gentamicin (10 µg), kanamycin (30 µg), linezolid (10 µg), mupirocin (200 µg), penicillin (1 U), tetracycline (30 µg), tobramycin (10 µg), and trimethoprim/sulfamethoxazole 135 (1.25/23.75 µg). The diameter of the inhibition zones was measured for each antibiotic disk and recorded in millimeters. The interpretation of results followed the recommendations given in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2019 guidelines with the exception of kanamycin that followed the Clinical and Laboratory Standards Institute (CLSI) 2017 recommendations. The minimal inhibitory concentrations (MICs) of vancomycin were determined by a standard broth microdilution method in sterile 96-well microplates according to the EUCAST guidelines. Briefly, bacterial suspension was adjusted to 0.5 McFarland standards and then diluted 1:20. Then, 50 µL of Mueller–Hinton broth, 50 µL of the antibiotic dilutions, and 5 µL of the inoculum were mixed and incubated at 37 ◦C for 24 h. Isolates showing a vancomycin MIC ≤ 4 µg/mL were considered susceptible and those showing an MIC > 4 µg/mL were classified as resistant. The reference strain *S. aureus* ATCC 25923 was used for quality control.

#### *4.3. DNA Extraction*

DNA extraction was performed as previously described. Briefly, 2 staphylococci colonies were suspended in 45 µL of Milli-Q water and 5 µL of lysostaphin (1 mg/mL) was added. The samples were incubated at 37 ◦C for 10 min, after which 45 µL of Milli-Q water, 150 µL of Tris-HCl (0.1 M) and 5 µL of proteinase K (2 mg/mL) were added. After 10 min of incubation at 67 ◦C, the samples were boiled at 100 ◦C for 5 min. The DNA was stored at −20 ◦C until use. The spectrophotometric quantification of DNA was carried out through the NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA) [55].

#### *4.4. Antimicrobial-Resistant Genes*

The presence of antimicrobial-resistant genes was investigated in each isolate according to the phenotypic resistance. The detection of the following antimicrobial-resistant genes was performed in a ProFlexTM PCR system (Applied Biosystems, Waltham, MA, USA): beta-lactams (*blaZ*, *mecA* and *mecC*), linezolid (*cfr*), aminoglycosides (*aac*(60 )*-*aph(200), *aph*(30 )-IIIa, *ant*(40 )-Ia and *str*), macrolides and lincosamide (*ermA*,*ermB*,*ermC*,*ermT*, *msr(A/B*), mphC, lnu*A*, lnu*B*, *vgaA and vgaB*), tetracycline (*tetK*, tet*M*, *tetL* and *tetO*), chloramphenicol (*fexA*, *fexB*, *catpC194*, *catpC221* and *catpC223*), fusidic acid (*fusB*, *fusC* and *fusD*) and trimethoprim/sulfamethoxazole (*dfrA*, *dfrG*, *dfrK* and *dfrD*). The protocol used for DNA amplification was as follows: a final volume of 50 µL contained 39.7 µL of ultra-pure water, 5 µL 10× complete buffer (Bioron, Römerberg, Germany), 1 µL 25 mM MgCl2, 1 µL deoxynucleotides triphosphate, 1 µL of each primer, 0.3 µL DFS Taq DNA polymerase (Bioron) and 1 µL DNA sample at 10 pg/µL. Primer sequences and PCR programs for the same are given in Table S2. The concentration and purity of the extracted DNA was measured using a spectrophotometer and Nano-DropTM software (Thermo ScientificTM, Waltham, MA, USA). Positive and negative controls used in all the experiments belonged to the strain collection of the University of Trás-os-Montes and Alto Douro.

#### *4.5. Statistical Analysis*

Pearson's chi-square test was used compare the carriage of *S. sciuri*, *S. lentus*, *S. urealyticus* and *S. haemolyticus* between the quails, the homebred chickens and the commercial chickens. The analyses were carried out using IBM SPSS Statistics, Version 26.0 (IBM Corp., Armonk, NY, USA) and significance was set at *p* ≤ 0.05.

#### **5. Conclusions**

MRCoNS are common bacteria found in healthy poultry in Portugal. *S. urealyticus* seems to be more prevalent in quails, while broiler chickens are more often colonized by *S. lentus*, indicating a separate epidemiology. The high frequency of MRCoNS isolates in this study may be due to the fact that these bacteria are colonizers of the normal skin flora of animals. However, the multidrug resistance found in almost all isolates indicates that MRCoNS in poultry may be an important reservoir of antimicrobial-resistant genes. This is of great concern for public health, since most antimicrobial resistances detected were antimicrobials commonly used in human medicine. Some measures to overcome antimicrobial resistance in poultry in Portugal should be taken into consideration, such as the education of poultry producers, limiting the availability of antibiotics and the application of strict legislation concerning antimicrobial prescription.

**Supplementary Materials:** The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/antibiotics11030365/s1: Table S1: Antimicrobial-resistant phenotype and genotype and SCC*mec* typing of CoNS isolated from poultry. Table S2: Primer pairs used for molecular typing and detection of antimicrobial resistance genes in MRSA strains. Figure S1: Prevalence of each staphylococci specie in poultry samples. References [56–71] are cited in the Supplementary Materials.

**Author Contributions:** Conceptualization, V.S., M.V.-P. and P.P.; methodology, V.S.; validation, V.S., M.C. and P.P.; investigation, V.S.; resources, M.V.-P. and C.S.; data curation, V.S. and E.F.; writing original draft preparation, V.S.; writing—review and editing, V.S., M.C. and P.P.; visualization, J.E.P.; supervision, J.L.C., G.I. and P.P.; funding acquisition, P.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the R&D Project CAREBIO2: Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics—towards innovative theranostic biomarkers, with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, financed by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020) and by the projects UIDB/CVT/00772/2020 and LA/P/0059/2020 funded by the Portuguese Foundation for Science and Technology (FCT). Vanessa Silva is grateful to FCT (Fundacão para a Ciência e a Tecnologia) for financial support through the PhD grant SFRH/BD/137947/2018.

**Institutional Review Board Statement:** The study was conducted according to the Helsinki Declaration (ICH-GCP principles), compliance with Schedule Y/ICMR Guidelines, the Oviedo Convention, and approved by the Ethics Committee of the University of Trás-os-Montes e Alto Douro (EC-UTAD, 8 November 2019).

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*2.3. Method*

#### 2.3.1. Synthesis of NaGdF4:49%Yb,1%Tm Core Nanocrystals

NaGdF4 doped with 49 mol % of Yb and 1 mol % of Tm (NaGdF4:49%Yb,1%Tm) was synthesized via a modified literature procedure [28,35,36]. A water solution of Gd(CH3CO2)3 (0.067 g; 0.2 mmol), Yb(CH3CO2)3 (0.069 g; 0.196 mmol), and Tm(CH3CO2)3 (0.001 g; 0.004 mmol) was combined with OA (5 mL) and ODE (5 mL) in a 50 mL two-neck round-bottom flask. The mixture was heated to 150 ◦C and maintained at this temperature for 1.5 h to form the lanthanide–oleate precursor. After cooling to 50 ◦C, a methanol solution consisting of NH4F (0.05 g; 1.36 mmol) and NaOH (0.04 g; 1 mmol) was added to the mixture and stirred for 30 min. The solution was heated to 100 ◦C for 20 min in vacuo to remove methanol. The resulting solution was quickly heated to 300 ◦C and maintained at this temperature for 1.5 h with nitrogen before cooling to room temperature. The obtained nanocrystals were precipitated by centrifugation at 8000 rpm for 5 min and then washed with cyclohexane and ethanol three times. The core nanoparticles were dispersed in cyclohexane (4 mL) for further shell coating.

#### 2.3.2. Synthesis of NaGdF4:49%Yb,1%Tm@NaYF4:20%Yb Core–Shell Nanocrystals

The synthesis procedure for core–shell nanoparticles was similar to that in our previous paper [36]. We use the obtained NaGdF4:49%Yb,1%Tm nanocrystals as seeds for subsequent shell coating. NaYF4 with 20 mol % of Yb (NaYF4:20%Yb) precursor was prepared via the same procedure as mentioned above, except that different amounts of OA (3 mL) and ODE (7 mL) were used. After cooling to 80 ◦C, the cyclohexane solution of NaGdF4:Yb/Tm nanoparticles (4 mL) was added and kept at 80 ◦C for 30 min to remove cyclohexane. Then, a methanol solution of NH4F (0.05 g; 1.36 mmol) and NaOH (0.04 g; 1 mmol) was added to the mixture and stirred at 50 ◦C for 30 min. Subsequently, the mixture was heated to 100 ◦C for 20 min in vacuo to remove methanol. The solution was then heated to 300 ◦C for 1.5 h under a nitrogen atmosphere. After cooling to room temperature, the core–shell nanoparticles were collected and washed using the same post-treatment approach as for core nanocrystals. NaGdF4@NaGdF4:49%Yb,1%Tm and NaYF4@NaGdF4:49%Yb,1%Tm were synthesized using a similar method to core–shell nanocrystals except for the use of NaGdF4 and NaYF4 as seeds.

2.3.3. Synthesis of NaGdF4:49%Yb,1%Tm@NaYF4:20%Yb@NaGdF4:50%Nd,10%Yb and NaGdF4:49%Yb,1%Tm@NaYF4:20%Yb@NaGdF4:50%Nd,10%Yb@NaGdF4 Core–Multishell Nanocrystals

The following multishelled core–shell nanoparticles were prepared using a procedure similar to the NaGdF4:49%Yb,1%Tm@NaYF4:20%Yb core–shell nanoparticles: NaGd F4@NaGdF4:49%Yb,1%Tm@ NaYF4:20%Yb; NaYF4@NaGdF4:49%Yb,1%Tm@NaYF4: 20%Yb; NaGdF4@NaGdF4:49%Yb, 1%Tm@NaYF4:20%Yb@NaGdF4:50%Nd,10%Yb; NaYF4 @NaGdF4: 49%Yb,1%Tm@NaYF4:20%Yb@NaGdF4:50%Nd,10%Yb;NaGdF4@NaGdF4:49%Yb,1%Tm@

NaYF4:20%Yb@NaGdF4:50%Nd,10%Yb@NaGdF4; NaYF4@NaGdF4:49%Yb,1% Tm@NaYF4: 20%Yb@ NaGdF4:50%Nd,10%Yb@NaGdF4.

#### 2.3.4. Preparation of Dye-Sensitized Upconversion Nanoparticles

The synthesis of IR-806 followed a well-established method [32]. Then, the IR-806 was dissolved in CHCl3 (0.01 mg/mL). The as-prepared core–multishell nanocrystals were centrifuged and dissolved in CHCl3 to a final concentration of 0.375 mg/mL. The samples were prepared by adding different amounts of IR-806 to Gd-CSYS2S3 CHCl3 solution (4 mL) and stirring for 2 h at a speed of 700 rpm at room temperature before UV– vis–NIR absorption and standard fluorescence measurements. All samples were prepared and measured in a dark environment.


*4.1. Single Target Immunoglobulin G (IgG) Probes*

Full-size IgG antibody probes have been applied to tumor detection, staging, guidance of local treatment, identification or validation of tumor targets, and assessment of therapeutic response or tumor prognosis [100]. Once the first-rank antigen has been selected, the corresponding IgG can be labeled with a radionuclide or fluorescent tag [19]. The radionuclide labeled IgG can be visualized via immunoPET imaging, and the fluorescent tags can be visualized through the fluorescence system during thyroidectomy or metastasectomy [101].


**6.3 Microstructure analysis**

512 Sintering of Ceramics – New Emerging Techniques

Fig. 9. X Ray Diffraction of equimolar mixture Al2O3 and TiO2 with concentrated placer

The quantification of Al2TiO5 formed, was determined by the internal standard method; the

AT+Additive (%) % %TiO2 unreacted % Al2O3 unreacted %Al2TiO5 formed

V2O5 6 20.1 25.5 54.4

MnO 6 11.2 14.1 74.7

FeTiO3 .SiO2 6 5.1 6.4 88.5 (mineral) 9 5.3 628 88.5

FeTiO3 6 2.3 2.8 94,8 (pure) 9 2.1 2.0 96,0

FeSi2 6 19.8 25.1 55.1

Table 2. Al2TiO5 % phase formation by sintering at 1450°C/3hours.

3 19.2 24.3 56.5

9 22.6 28.7 48.7 3 13.1 16.6 70.4

9 9.4 11.9 78.7 3 5.5 5.8 88,7

3 1.9 2.7 95,0

3 21.3 27.1 51.7

9 16.9 21.5 61.6

ilmenite (FeTiO3.SiO2): 3, 6 and 9 wt% addition, sintered at 1450°C for 3 hours.

**6.2.1 Al2TiO5 formation phase quantification**

achieved results are showed in Table 2.

The composition without addition (Fig. 10), shows the characteristic microstructure of the aluminum titanate: a porous and microcracked Al2TiO5 matrix phase and the presence of unreacted Al2O3 and TiO2, due to the formation reaction kinetics, which is a process leaded by nucleation and growth of Al2TiO5 grains and finally the diffusion of the reactants remnants through the matrix, this is controlled for a very slow reacting species diffusion, as it was found by: Wohlfromm et al., (1991).

Fig. 10. a)BSE microstructure of Al2O3 and TiO2 without addition, sintered at 1450°C for 3 hours, b) EDS of the matrix with an exact atomic relationship: 25 at% Al, 12.5 at% Ti and 62.5 at% O. (AT: Aluminum titanate; Ti: Titania; Al: Alumina).

The addition of the low melting point V2O5 (678°C) is evidenced in the microstructure with the presence of an abundant glassy intergranular phase, which constitutes a physical barrier between Al2O3 and TiO2, retarding the Al2TiO5 formation (Fig. 11a).

Fig. 11. a)BSE microstructure detail of Al2O3 and TiO2 with V2O5: 6 wt% addition, sintered at 1450°C for 3 hours. b) EDS of the intergranular glassy phase (GP), appearing due to V2O5 low melting point. (AT: Aluminum titanate; Ti: Titania; Al: Alumina).

Reactive Sintering of Aluminum Titanate 515

**Al**

**Si**

**Au Rec.**

Fig. 13. c) EDS of ternary eutectic (TE) and d) EDS of the intergranular Fe rich phase (FeP).

2011).

**O**

**Al**

**Si**

practically free of unreacted original phases.

**0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00**

Element Wt% At% Ok 32.68 46.66 AlK 37.26 31.54 SiK 22.18 18.04 TiK 7.89 3.76 Total 100.00 100.00

**Au Rec.**

**Ti**

A fraction of free Al2O3 remains as an intragranular phase, the TiO2 reacts completely and besides the Al2TiO5 matrix phase a ternary eutectic reaction grainy phase is formed between the Al2O3-SiO2-TiO2 (as low as it is undetected by RXD), but increasing its quantity with the additive and, also a fourth intergranular phase rich in Fe is depicted (Fig.13d) (Arenas et al.

**O**

**1.10 2.00 2.90 3.80 4.70 5.60 6.50 7.40**

**Ti**

Element Wt% At% Ok 28.81 47.95 AlK 25.75 25.41 SiK 7.96 7.55 TiK 15.35 8.53 FeK 22.14 10.56 Total 100.00 100.00

**c) d)**

**TE FeP**

**Fe**

**AT**

Pure and concentrated ilmenite (FeTiO3), additions have a beneficial effect on grain growth control (Fig. 14 and 15). The SiO2 left in the purified mineral promoted the formation of an intergranular liquid phase which could not be detected by XRD. Microstructures are

Fig. 14. a) BSE microstructural x1000 and a detail X3000, of Al2O3 and TiO2 with pure FeTiO3: 6 wt% addition, sintered at 1450°C for 3 hours. Notice the grain growth control.

Althought localized EDS analysis on the glassy phase was carried out, the Al and Ti values obtained are due to the larger electron beam action volume compared to phase size (Fig. 11b).

The microstructure of MnO added samples shows extensive Al2TiO5 phase formation, with a minor presence of liquid phase, product of the two eutectic reactions, at 1290°C and 1330°C, between MnO and TiO2. However, opposite to the V2O5 added samples, the reacting species diffusion and Al2TiO5 formation is accelerated with the MnO contents and, unreacted TiO2 is absent in the microstructure, due to the secondary reactions. EDS analysis identified the intergranular eutectic phase as 2MnO.TiO2 and MnO.TiO2 (Fig. 12). Microstructure grain size decreased with MnO contents in the sintered bodies.

Fig. 12. a)BSE microstructural detail of Al2O3 and TiO2 with MnO: 6 wt% addition, sintered at 1450°C for 3 hours. b) EDS of the intergranular MnTiO3 eutectic phase (EP). (AT: Aluminum titanate; Al: Alumina).

The FeSi2.Si modified composition has a different microstructure to that obtained with other additives (Fig.13a-d).

Fig. 13. a) BSE microstructural detail of Al2O3 and TiO2 with FeSi2.Si: 6 wt% addition, sintered at 1450°C for 3 hours. b)EDS of the Al2TiO5 matrix.

Althought localized EDS analysis on the glassy phase was carried out, the Al and Ti values obtained are due to the larger electron beam action volume compared to phase size (Fig.

The microstructure of MnO added samples shows extensive Al2TiO5 phase formation, with a minor presence of liquid phase, product of the two eutectic reactions, at 1290°C and 1330°C, between MnO and TiO2. However, opposite to the V2O5 added samples, the reacting species diffusion and Al2TiO5 formation is accelerated with the MnO contents and, unreacted TiO2 is absent in the microstructure, due to the secondary reactions. EDS analysis identified the intergranular eutectic phase as 2MnO.TiO2 and MnO.TiO2 (Fig. 12).

Fig. 12. a)BSE microstructural detail of Al2O3 and TiO2 with MnO: 6 wt% addition, sintered

**Al**

**Au Rec .**

**Al**

**1.40 2.00 2.60 3.20 3.80 4.40 5.00 5.60 6.20**

**a) b)**

Element Wt% At% Ok 35.14 54.24 AlK 30.83 28.22 TiK 34.02 17.54 Total 100.00 100.00

**Au Rec .**

**1.00 1.60 2.20 2.80 3.40 4.00 4.60 5.20 5.80**

**Ti**

**AT**

**Mn**

**a) b)**

**Ti** Element Wt% At% Ok 12.43 28.26 AlK 13.48 18.18 TiK 45.89 34.36 MnK 28.21 18.69 Total 100.00 100.00

**EP EP**

The FeSi2.Si modified composition has a different microstructure to that obtained with other

Fig. 13. a) BSE microstructural detail of Al2O3 and TiO2 with FeSi2.Si: 6 wt% addition,

**O**

sintered at 1450°C for 3 hours. b)EDS of the Al2TiO5 matrix.

**Al**

**FeP**

at 1450°C for 3 hours. b) EDS of the intergranular MnTiO3 eutectic phase (EP).

**AT**

(AT: Aluminum titanate; Al: Alumina).

**AT**

**Al**

additives (Fig.13a-d).

**TE**

Microstructure grain size decreased with MnO contents in the sintered bodies.

11b).

Fig. 13. c) EDS of ternary eutectic (TE) and d) EDS of the intergranular Fe rich phase (FeP).

A fraction of free Al2O3 remains as an intragranular phase, the TiO2 reacts completely and besides the Al2TiO5 matrix phase a ternary eutectic reaction grainy phase is formed between the Al2O3-SiO2-TiO2 (as low as it is undetected by RXD), but increasing its quantity with the additive and, also a fourth intergranular phase rich in Fe is depicted (Fig.13d) (Arenas et al. 2011).

Pure and concentrated ilmenite (FeTiO3), additions have a beneficial effect on grain growth control (Fig. 14 and 15). The SiO2 left in the purified mineral promoted the formation of an intergranular liquid phase which could not be detected by XRD. Microstructures are practically free of unreacted original phases.

Fig. 14. a) BSE microstructural x1000 and a detail X3000, of Al2O3 and TiO2 with pure FeTiO3: 6 wt% addition, sintered at 1450°C for 3 hours. Notice the grain growth control.

Reactive Sintering of Aluminum Titanate 517

For FeSi2.Si added samples (Fig.17), although the presence of oxidizing atmosphere, leads to the oxidation of Si and subsequent formation of ternary liquid phase between Al2O3, TiO2 and SiO2 which promotes a good densification, it has a minimal beneficial effect on stabilization. The presence of Al2TiO5 diffraction peaks is small if compared with that of Al2O3 and TiO2, product of decomposition. It might be explained as, that only a fraction of

Al2O3 TiO2 Al2TiO5

20 30 40 50 60 70

Fig. 17. X Ray Diffraction of Al2TiO5 with FeSi2.Si 6 wt% addition, heat treated for 100hours

The addition of pure FeTiO3 (ilmenite*)* clearly shows an increase in the aluminum titanate

Al2O3 TiO2 Al2TiO5

20 30 40 50 60 70

Fig. 18. X Ray Diffraction of Al2TiO5 with FeTiO3 6wt% addition, heat treated for 100hours at

2 theta

2 theta

6% FeTiO3

6% FeSi2.Si

the Fe ions from the FeSi2 react and substitute the Al+3 ions, stabilizing the material.

HT 100h 1100ºC

Intensity (A.U)

at 1100°C.

1100°C.

stabilization (Fig. 18).

HT 100h 1100ºC

Intensity (A.U)

Fig. 15. a) BSE microstructural detail X3000, of Al2O3 and TiO2 with with concentrated placer ilmenite (FeTiO3. SiO2): 6 wt% addition, sintered at 1450°C for 3 hours. b) EDS of intergranular phase due to SiO2 presence.

#### **6.4 Thermal stability**

In order to determine the compositions stability, XRD analyses were performed on samples heat treated at 1100°C for 100 hours. The temperature selection is based on industrial applications working conditions and the maximum temperature for decomposition to occur.

The samples without additives and those with V2O5 and MnO showed a complete decomposition after heat treatment, as only the diffraction peaks of Al2O3 and TiO2 were showed. (Fig. 16).

Fig. 16. X Ray Diffraction Al2TiO5 without addition, with 6wt%V2O5 and 6wt% MnO, heat treated for 100hours at 1100°C.

**a)**

Fig. 15. a) BSE microstructural detail X3000, of Al2O3 and TiO2 with with concentrated placer

**O**

In order to determine the compositions stability, XRD analyses were performed on samples heat treated at 1100°C for 100 hours. The temperature selection is based on industrial applications working conditions and the maximum temperature for

The samples without additives and those with V2O5 and MnO showed a complete decomposition after heat treatment, as only the diffraction peaks of Al2O3 and TiO2 were

No addition HT 100h

Al2O3 TiO2 Al2TiO5

20 30 40 50 60 70

Fig. 16. X Ray Diffraction Al2TiO5 without addition, with 6wt%V2O5 and 6wt% MnO, heat

2 theta

6% V205

Element Wt% At% Ok 17.61 32.28 AlK 33.60 36.53 SiK 3.02 3.15 TiK 45.78 28.04 **Al** Total 100.00 100.00

**Au Rec . Si**

**Ti**

**IP**

**b)**

6% Mn0

ilmenite (FeTiO3. SiO2): 6 wt% addition, sintered at 1450°C for 3 hours. b) EDS of

intergranular phase due to SiO2 presence.

**AT**

1100ºC

Intensity (A.U)

treated for 100hours at 1100°C.

**6.4 Thermal stability**

decomposition to occur.

showed. (Fig. 16).

For FeSi2.Si added samples (Fig.17), although the presence of oxidizing atmosphere, leads to the oxidation of Si and subsequent formation of ternary liquid phase between Al2O3, TiO2 and SiO2 which promotes a good densification, it has a minimal beneficial effect on stabilization. The presence of Al2TiO5 diffraction peaks is small if compared with that of Al2O3 and TiO2, product of decomposition. It might be explained as, that only a fraction of the Fe ions from the FeSi2 react and substitute the Al+3 ions, stabilizing the material.

Fig. 17. X Ray Diffraction of Al2TiO5 with FeSi2.Si 6 wt% addition, heat treated for 100hours at 1100°C.

The addition of pure FeTiO3 (ilmenite*)* clearly shows an increase in the aluminum titanate stabilization (Fig. 18).

Fig. 18. X Ray Diffraction of Al2TiO5 with FeTiO3 6wt% addition, heat treated for 100hours at 1100°C.

Reactive Sintering of Aluminum Titanate 519

**Ti**

**Al**

**a) b)**

**c) d)**

**e)**

Fig. 20. a) BSE Microstructural detail x3000, Al2TiO5 with : a) V2O5 6wt% ; b) MnO 6wt%; c) FeSi2.SiO2 6wt% d) FeTiO3 6wt% and e) FeTiO3.SiO2 9wt% addition, heat treated at 1100°C

**AT**

The quantification of Al2TiO5 decomposed after heat treatment was also determined using the internal standard method being the results showed in Table 3. The values obtained corroborate BSE image analysis, observations. The stabilization addition effect being higher in the compositions with pure and concentrated mineral, the MnO and the ferrosilicon

for 100 hours. (AT: Aluminum titanate; Ti: Titania; Al: Alumina).

stabilize slightly while vanadium oxide have not any effect.

**6.5 Al2TiO5 decomposition phase quantification**

**Ti**

**Al AT**

**Al**

**Ti**

**Al**

**Ti**

This behavior agrees with the expected solid solution formation between FeTiO3 and Al2TiO5, as depicted in the calorimetric studies (DSC). These experiments show the decomposition of ilmenite in air atmosphere to Fe2O3 and TiO2 with the formation of Fe2TiO5 (Suresh et al.,1991), followed by Al2TiO5 reaction at higher temperature. This allows the possibility of a solid solution formation between Al2TiO5 and the isostructural Fe2TiO5 by a cation replacement mechanism.

From the ionic radii concept, the structural stabilization might be explained by the incorporation of Fe+ 3 (r=0.67Å) which decreases the structure distortion, caused by the Ti+ 4: Al+ 3 radii difference (Shannon, R., 1969).

In the case of ilmenite addition, but from the concentrated mineral (Fig.19), the structural stabilization effect is evidenced only for the composition with 9% addition. This behavior might be attributed to the SiO2 contamination which, on the other hand, benefits body densification by a liquid phase sintering mechanism.

Fig. 19. X Ray Diffraction of Al2TiO5 with FeTiO3.SiO2 9wt% addition, heat treated for 100hours at 1100°C.

The microstructure study by SEM-EDS corroborated the X-ray analysis evaluation (Fig. 20). All heat treated samples showed the characteristic elongated grain shape, typical of the Al2TiO5 decomposition into its original precursors raw materials Al2O3 and TiO2. However, stabilized Al2TiO5 phase is observed in the compositions with 6% pure ilmenite, 9% concentrated mineral and in lower proportion in the samples with 6% ferrosilicon.

This behavior agrees with the expected solid solution formation between FeTiO3 and Al2TiO5, as depicted in the calorimetric studies (DSC). These experiments show the decomposition of ilmenite in air atmosphere to Fe2O3 and TiO2 with the formation of Fe2TiO5 (Suresh et al.,1991), followed by Al2TiO5 reaction at higher temperature. This allows the possibility of a solid solution formation between Al2TiO5 and the isostructural Fe2TiO5

From the ionic radii concept, the structural stabilization might be explained by the incorporation of Fe+ 3 (r=0.67Å) which decreases the structure distortion, caused by the Ti+ 4:

In the case of ilmenite addition, but from the concentrated mineral (Fig.19), the structural stabilization effect is evidenced only for the composition with 9% addition. This behavior might be attributed to the SiO2 contamination which, on the other hand, benefits body

Al2O3 lTiO2 Al2TiO5

20 30 40 50 60 70

The microstructure study by SEM-EDS corroborated the X-ray analysis evaluation (Fig. 20). All heat treated samples showed the characteristic elongated grain shape, typical of the Al2TiO5 decomposition into its original precursors raw materials Al2O3 and TiO2. However, stabilized Al2TiO5 phase is observed in the compositions with 6% pure ilmenite, 9%

Fig. 19. X Ray Diffraction of Al2TiO5 with FeTiO3.SiO2 9wt% addition, heat treated for

concentrated mineral and in lower proportion in the samples with 6% ferrosilicon.

2 theta

9% FeTiO3.SiO2.

.

by a cation replacement mechanism.

HT 100h 1100ºC

Intensity

100hours at 1100°C.

 (A.U)

Al+ 3 radii difference (Shannon, R., 1969).

densification by a liquid phase sintering mechanism.

Fig. 20. a) BSE Microstructural detail x3000, Al2TiO5 with : a) V2O5 6wt% ; b) MnO 6wt%; c) FeSi2.SiO2 6wt% d) FeTiO3 6wt% and e) FeTiO3.SiO2 9wt% addition, heat treated at 1100°C for 100 hours. (AT: Aluminum titanate; Ti: Titania; Al: Alumina).

### **6.5 Al2TiO5 decomposition phase quantification**

The quantification of Al2TiO5 decomposed after heat treatment was also determined using the internal standard method being the results showed in Table 3. The values obtained corroborate BSE image analysis, observations. The stabilization addition effect being higher in the compositions with pure and concentrated mineral, the MnO and the ferrosilicon stabilize slightly while vanadium oxide have not any effect.

Reactive Sintering of Aluminum Titanate 521

It has confirmed the presence of the Fe+3 ions in all compositions with Fe added, i.e., the

The Mössbauer spectrum for material with 6% of ferrosilicon addition can be adjusted to two doublets (Fig.21a). The first doublet corresponds to the ferrous cation (Fe+2) with a resonance that fits the hyperfine splitting with an isomer shift: IS = 1.01 ± 0.002 mm/s and a quadrupole splitting: QS= 0.664 ± 0.003 mm/s. The second doublet corresponds to the resonance of the ferric cation (Fe+3) with a IS = 0. 323 ± 0.003 mm/s and a QS = 0. 520 ± 0.004 mm/s. For composition with 6% pure ilmenite addition (Fig. 21b.), it is revealed a consistent doublet with the ferric state (Fe+3), with a IS = 0. 323 + 0.003 mm/s and QS = 0.520 ± 0.004 mm/s. The spectrum for the sample with 6% of mineral ilmenite (Fig. 19c), one could guess the doublet corresponds to both states ferrous (Fe+2) and ferric (Fe+3), however should be

Velocity (mm/s

Relative Absortion

 (%) 0.0

Velocity (mm/s

**a)** 0.9 **b)**

**c)**

Fig. 21. Mössbauer Spectroscopy of Al2TiO5 with: a) 6wt% FeSi2.SiO2 ; b) 6wt% FeTiO3 and

These results corroborate the possible replacement of the Al+3 ions by ion Fe+3; there is higher stabilization in samples with pure ilmenite addition, where all Fe ions are in ferric state. However, as it was found in previous research (Barrios de Arenas & Cho, 2010) , the presence of Fe+2 ions, also represent the possibility of Al+3 ions substitution, with the

creation of defects, which in turn promotes the diffusion in solid state.

noted that results are not accurate, with considerable dispersion.

Velocity (mm/s

 (%)

0.0

0.9

Relative Absortion

c) 6wt% FeTiO3.SiO2 additions.

**6.7 Mössbauer spectroscopy**

ilmenites and ferrosilicon.

Relative Absortion

0.9

 (%) 0.0

Table 3. Al2TiO5 % Phase decomposition after heat treatment at 1100°C/ 100hours, by internal standard quantification method.


*4.3. Celestial Bodies*

#### 4.3.1. Moon

The moon and its various phases played an important role in the profane aspects of ancient Near East life in the marking of time. The moon also enjoyed a lively sacred history as a part of ritual calendars and in association with a variety of lunar deities. Deification and worship of the moon is attested across the ancient Near East, most famously at the Mesopotamian sites of Ur and Harran (by the names Nanna and Suen [=Sîn] respectively), where traditions of moon-god worship span several millennia (Green 1992; Margueron 1992). In the West Semitic sphere, second and first millennium epigraphic texts, mid-first millennium biblical texts, and a variety of iconographic material underscore the importance of the moon and the moon-god, whose name is usually derived from the West Semitic word for the moon, *yrh.* (Schmidt 1999; Theuer 2000). In Ammon, there are a handful of items that make it clear that the West Semitic moon-god played an important role in Ammonite perception of nonphysical beings.

On the epigraphic side of things, two bits of evidence have come to light. A small statue, now generally called the Statue of Yarh.ގazar (*CAI*, no. 43), was found outside the Roman city wall on the north end of the Amman Citadel. The statue is carved from limestone and stands 45 cm high, and was found with traces of red paint on the body and garments (Barnett 1951, pp. 34–35, and pl. XI). Because the statue was not found in a secure archaeological context, it is usually dated to the late eighth or seventh century BCE, based on the script of the inscription on the pedestal base (*CAI*, p. 108). The partially preserved two-line inscription reads:

Line 1: [broken]*šwyrh.*ގ*zr* [broken]*šw* Yarh.ގazar Line 2: [broken]*kr br šnb*14 [broken]*kr* son of Šanib

Despite the broken parts of the inscription, it appears to preserve a chain of three generations with only the last two letters (*kr*) of the middle generation being visible. Reconstructed, the names would most likely run as such: "Yarh.ގazar, [son of ?]*kr*, son of Šanib."

14 This has been read as either a "b" or "p" (*CAI*, p. 210).

The grandfather mentioned at the end of the inscription is plausibly identified with "Šanipu of the House of Ammon," a king mentioned as paying tribute to the Neo-Assyrian monarch Tiglath-pileser III in 734 BCE (**?**, pp. 122–23, no. 47 [Summ. 7r. 10']B114-religions-432110) Whether this means that Šanipu's son and grandson were kings too, one cannot say for sure, but they were certainly related to one of the power-players in Ammon. Whatever Yarh.ގazar's specific status, his name includes the name of the West Semitic moon-god Yarah. , and means "Moon is help." As a representation of what must have been a member of the Ammonite elite, the inscription shows that the West Semitic moon-god had a meaningful place in the Ammonite pantheon.

Apart from the statue of Yarh.ގazar, only one other plausibly Ammonite inscription contains the *yarah.* element. A badly burnt ostracon from Tall al-Mazar, Ostracon IV, has only one partially legible word that appears to read *lyrh.* , "to Yarah. ." This could represent a sacrifice to the moon-god or something given to a person with the same name (*CAI* no. 145). The poor state of preservation of the ostracon precludes certainty.

Iconographic representations of the moon from the region of Ammon also attest to the worship of the moon-god. Two nearly identical architectural examples come from the site of Rujm al-Kursi, about 10 km west of the center of Amman, Jordan. There, on either side of the entrance to a building that is plausibly identified as a temple, are smoothed, nearly cubical blocks of limestone measuring slightly less than 1.0 m per side. On the front face of each block there is a relief presenting a lunar crescent standing on a table and supported by a pillar (Figure 3).15 Inside the crescent there is a disk which may represent the full moon (Keel 1998, p. 113). The pillar on the better-preserved block shown in Figure 3 has two rectangles inside it. The pedestal or platform on which the pillar rests has feet rather than the solid or stepped pedestals known on other stelae and seals. Inside the table are two crossing lines that look like an 'X.' The iconography of these reliefs is similar to the iconography of the crescent moon standard typical of the Mesopotamian moon-god Sîn of Harran. The main exception is that the standards here lack the tassels that are typical of the lunar crescent of Sîn of Harran (Hübner 2009, p. 150), but stelae depicting the crescent standard without the tassels are known, even from areas closer to Harran (Keel 1998, pp. 63–64, figs. 3–4). As Keel points out, the thickened pillar, and the style of the platform have parallels on seals from the region (Keel 1998, p. 113, especially figs. 36–37). One wonders whether the 'X' inside the platform is a simplified version of the more extensive cross-hatching visible on some seals (Keel 1998, figs. 33–34, 38–39, 42–49). While a full analysis of this site must await the final publication of the excavations, the reliefs have been dated on stylistic grounds to roughly the seventh century BCE, at the height of Assyrian power in the region. If the date is confirmed by the pottery, these reliefs will furnish evidence of the spread of the iconography of Sîn of Harran well into the Assyrian periphery. Whether this indicates the worship of Sîn of Harran in Ammon, or the adaptation of Sîn's iconography for application to the local moon-god Yarah. is difficult to determine (Hübner 2009, pp. 149–50). Given the relative popularity of the moon-god among the Arameans in Syria, including stelae with moon-god iconography at Bethsaida and the Hauran to the northwest and north of Ammon (Bernett and Keel 1998; Keel 1998, pp. 116–20), moon-god traditions from Harran were most likely mediated through the Arameans.16

15 The Iron Age material from the site has not been published by the excavator, Abdel-Jalil ގAmr, who has since died. A brief note from ގAmr mentions Iron Age material in deep layers (Amr 1986). The material published from the site comes from the Roman, Byzantine, and Islamic periods. For bibliography see (Hübner 1992, p. 152, n. 121). The possible Iron Age temple and its reliefs were first discussed by Keel, who had been informed of the site by Ulrich Hübner (Keel 1998). Hübner later published an article on the site (Hübner 2009). Subsequent discussion of the possible temple can be found in (Elkowicz 2012, pp. 90, 114, Taf. 62, 66; Elkowicz 2014; Hübner 2015; Tyson 2014, pp. 37–39). The drawing from Keel (1998, fig. 105) was reproduced and mentioned briefly in Staubli (2003, p. 67).

16 Aramean cultural influence on Ammon can be seen in other ways, for example, the adoption of the Aramaic script in Ammon sometime in the late ninth or early eighth centuries BCE (Tyson 2014, pp. 102–4), and artistic influences visible in Ammonite sculpture from the eighth century BCE (Dornemann 1983, pp. 156–59; Zayadine 1991, pp. 49–51).

**Figure 3.** Lunar crescent relief from one side of the entrance to the Rujm al-Kursi building. Photo courtesy of the author.

The images on seals from Ammon likewise contribute to the evidence for the moon-god. Three main categories of seals are typical of the Iron Age II in Syro-Palestine and are based on shape: cylinder seals, conoid stamp seals, and scaraboid stamp seals.17 By all accounts, the scaraboid stamp seals represent the indigenous tradition of sealing. The cylinder seals that are somewhat common in Syro-Palestine during the Neo-Assyrian Period (734–612 BCE), and the conoid stamp seals that become more common in Syro-Palestine in the Neo-Babylonian and first part of the Persian Period (ca. 600–450 BCE), have their home in Mesopotamia (Keel and Uehlinger 1998, pp. 367, 374; Ornan 1993, pp. 52–53).18 As such, it is not always clear whether cylinder seals and conoid stamp seals found in Syro-Palestine represent local perceptions of the divine world. However, contemporary parallels in the iconography of the three main types suggest convergences of foreign and indigenous perceptions of the divine world.

Two cylinder seals from the region depict the standard of Sîn of Harran. On Nebo 1 (*CSAJ*, pp. 192–93),19 which dates to the 8th–7th centuries BCE, two worshippers approach a tasseled crescent moon standard of Sîn of Harran (Figure 4a). The worshippers carry what are probably instruments, and are separated by ankh symbols. On the opposite side of the crescent moon standard is a stylized tree. The seal also has other astral symbols, including the eight-rayed star of Venus, and three dots, likely representing stars (*CSAJ*, p. 192; Keel and Uehlinger 1998, p. 302). Another cylinder seal dating to the eighth–seventh centuries BCE (*CSAJ*, pp. 306–7, Tall al-Mazar 23) shows two falcon-headed figures, which attend the tasseled standard of Sîn of Harran (Figure 4b). The scene also contains the eight-rayed star of Venus and a schematic, seven-dot representation of the Pleiades. On the outside of each of the figures are what seem to be stylized trees.20 An additional exemplar (*CSAJ*, pp. 356–57, Tall al-ގUmeiri 78) with a less clear image appears to portray a crescent moon standard, along with other astral symbols (Figure 4c). While the iconography on these cylinder seals probably does not arise from local sources, it does illustrate the full repertoire of symbols and figures that could appear in such a scene. On stamp seals, which have less surface area, one tends to find more limited representations of worship scenes.

17 The term "scaraboid" as a description of the slightly oblong shape of these seals, comes from scarab seals, which have scarab beetles inscribed on the convex side of the seal.

18 The shape of a seal affects the amount of space for images and the way in which the seal is used. Cylinder seals are typically carved all the way around the cylinder, and when used for sealing, they create an image that repeats if rolled more than one rotation. Conoid and scaraboid stamp seals on the other hand, have a single main, roundish stamping surface that has a smaller space than what is typical on a cylinder seal, hence limiting what can be carved on it.

19 *CSAJ* = Eggler, Jürg, and Othmar Keel. 2006. *Corpus der Siegel-Amulette aus Jordanien vom Neolithikum bis zur Perserzeit*. Fribourg: Academic Press.

20 Two other cylinder seals that were purchased in Amman, and are hence not from a secure archaeological context, appear to have a crescent moon along with other astral symbols (*CSAJ*, pp. 58–59, ގAmman 87–88).

**Figure 4.** Cylinder seals with crescent moon standards: (**a**) Nebo 1; (**b**) Tall al-Mazar 23; (**c**) Tall al-ގUmeiri 78.

A number of stamp seals from regions around Ammon depict the standard of Sîn of Harran. The elements that flank the standard of Sîn of Harran can be humans, plants, or some combination of the two (Keel and Uehlinger 1998, pp. 304–6). The total scene remains relatively stable, but the flanking figures alternate between a single person, two people, or a person and a stylized tree. One of the seals of this group seems to take the scene in yet another way. It appears to represent the deity in anthropomorphic form flanked by two stylized trees. This is arguably the moon-god, because the scene follows the pattern, and the figure is sitting on top of a low table or bench that is parallel to the bench on which the standard of Sîn of Harran usually sits (Keel and Uehlinger 1998, pp. 306–7).

In Ammon, two main variations appear on stamp seals. The first includes a worshipper with an upraised hand standing in front of several cult symbols, including a crescent moon. Four conoid stamp seals follow this pattern. In two of them with very similar styles from the 7th–6th centuries BCE (ގAmman 3, Meqabelein 1; Figure 5a,b), there is a worshipper standing with their hand raised before a platform that houses the symbols of two Mesopotamian deities: the stylus of Nabu and the spade of Marduk. Above stands the crescent moon (*CSAJ*, pp. 10–11, 190–91). These scenes clearly emphasize the roles of Marduk and Nabu, but at the same time, they include the crescent moon. Two conoid stamps from Tall al-Mazar display worshippers before cultic symbols (Figure 5c,d). In the case of Tall al-Mazar 1 (sixth century BCE), the image shows a worshipper with their hand upraised before the lamp of Nusku, with a crescent moon at the top of the scene (*CSAJ*, pp. 296–97). A relatively minor god for much of Mesopotamian history, Nusku became more important in Neo-Assyrian times through an association with Sîn of Harran (Black and Green 1992, p. 145). Tall al-Mazar 14 (seventh century BCE) has two worshippers standing in front of the symbols of Nabu and Marduk. The top of the scene is filled out by astral symbols: the eight-rayed star of Venus, the crescent moon, and the winged sun disk (*CSAJ*, pp. 300–1).

**Figure 5.** Stamp seals with worshipper and crescent moon: (**a**) ގAmman 3; (**b**) Meqabelein 1; (**c**) Tall al-Mazar 1; (**d**) Tall al-Mazar 14; (**e**) Tall al-ގUmeiri 51; (**f**) Tall al-ގUmeiri 52; (**g**) Umm Udeina 5.

Several scaraboid stamp seals also display a worshipper with a crescent moon. The best-preserved example is Tall al-ގUmeiri 52 (8th–7th centuries BCE; Figure 5f). There is a worshipper—whether human or not is hard to tell—with both hands out towards a crescent moon with a full moon inside. In front of the figure is a branch or stylized tree (*CSAJ*, pp. 340–41). The limits of space on the seal mean much of the larger scene could not be included. The seal cutter retained the moon and the tree or branch that would otherwise flank the scene. Two others (Tall al-ގUmeiri 51; Umm Udeina 5;

Figure 5e,g) seem to preserve a similar scene with a worshipper before a crescent moon, but both are worn and somewhat difficult to decipher (*CSAJ*, pp. 340–41, 476–77).21

The second main variation includes an animal along with a crescent moon, which often also has a full moon inside it. Two conoid stamp seals fit this variation. ގAmman 4 (Figure 6a), which comes from the 6th–5th centuries, depicts a sphinx-like animal with what is perhaps a stylized ankh, as well as a crescent moon above (*CSAJ*, pp. 10–11). Tall al-ގUmeiri 66 (Figure 6b), a seal from the seventh century, displays a quadruped with horns bending forward, a nursing child, and a very schematic set of celestial imagery above including what appears to be a lunar crescent (*CSAJ*, pp. 348–49). Several scaraboid stamp seals also fit this variation. Hisban 2, Tall al-Mazar 18, and Tall al-ގUmeiri 11, and 54 all pair quadrupeds with a crescent moon (Figure 6c–f). On Tall al-Mazar 18 and Tall al-ގUmeiri 11 the animals appear to be bovine, with bodies carved in three segments. Horns are clearly visible on the two bovines on Tall al-ގUmeiri 11, while they are less clear on Tall al-Mazar 18. The exact animal on Hisban 2 and Tall al-ގUmeiri 54 is a bit more difficult to discern, though both have horns curved backwards. All but Tall al-ގUmeiri 54 contain a full moon inside the crescent moon (*CSAJ*, pp. 168–69, 302–3, 318–19, 340–41). Tall al-ގUmeiri 4 (Figure 6g) is a seal impression on clay dated to the early sixth c. BCE, which combines lunar symbols with a scarab beetle (*CSAJ*, pp. 312–13; = *CAI*, no. 129). Flanking the beetle are two standards with a disc (perhaps a full moon) above which are downward facing lunar crescents.

**Figure 6.** Seals with an animal and crescent moon: (**a**) ގAmman 4; (**b**) Tall al-ގUmeiri 66; (**c**) Hisban 2; (**d**) Tall al-Mazar 18; (**e**) Tall al-ގUmeiri 11; (**f**) Tall al-ގUmeiri 54; (**g**) Tall al-ގUmeiri 4.

A number of stamp seals have astral symbols without any other attending imagery. The badly damaged conoid stamp seal ގAmman 53 (seventh–sixth centuries BCE; Figure 7a) shows the crescent moon and the eight-rayed star of Venus (*CSAJ*, pp. 40–41).22 The scaraboid stamps seals Tall al-ގUmeiri 39 (seventh–sixth centuries BCE; Figure 7b) and Umm Udeina 1 (eighth century BCE; Figure 7c) are both largely epigraphic, but ގUmeiri 39 has what appears to be a crescent moon with full moon at the bottom, and Umm Udeina 1 has a crescent moon and star (*CSAJ*, pp. 332–33, 474–75).

21 Cf. also the scaraboid seal Tall Deir ގAlla 28 from the nearby site of Tall Deir ގAlla (*CSAJ*, pp. 406–7). 22 Cf. Tall as-Sa'idiya 8 (*CSAJ*, pp. 370–71).

**Figure 7.** Seals with astral symbols: (**a**) ގAmman 53; (**b**) Tall al-ގUmeiri 39; (**c**) Umm Udeina 1; (**d**) 5. Ammanގ

A variation of Sîn iconography that is common in Assyria and the northern part of Syria between the ninth and fifth centuries BCE is an anthropomorphic deity standing in a crescent moon with a hand raised in a blessing posture (Keel and Uehlinger 1998, p. 309). A conoid stamp seal from Amman (ގAmman 5) contains just such an image (Figure 7d). The seal, which probably dates to the seventh century BCE, has a deity standing in a crescent moon blessing a worshipper (*CSAJ*, pp. 12–13).23

The varied evidence available for the moon-god in Ammon—epigraphic and iconographic demonstrates the active role that the moon-god played in Ammonite society. The cylinder seals that portray the standard of Sîn were most likely imported or came to Ammon with a trader or official from Assyria. The depiction of the lunar crescent on the six conoid stamp seals discussed above appears invariably alongside the symbols for other identifiably Mesopotamian gods (Marduk, Nabu, Nusku). Since the tradition of conoid stamp seals generally emanates from Syria and Assyria, it is possible to regard them as imports similar to the cylinder seals. The scaraboid stamp seals are, on the other hand, a distinctly Levantine tradition. The exemplars from Ammon preserve three main variants: a worshipper before a lunar emblem, a lunar emblem with an animal (bovine or other quadruped), and a lunar emblem with an inscription (in one case also including a star). Absent from the scaraboid stamp seals are Sîn's standard and the symbols of other Mesopotamian gods, and this fact makes the scaraboid stamp seals the most potentially polyvalent of the three types of seals found in Ammon. A crescent moon is a nearly universal emblem of moon-gods across the ancient Near East and thus could invoke any locally or more broadly known moon-god. Is there a way then to specify which moon-god is referenced in a specific context such as Ammon?

Given the occurrence of Yarah. as a theophoric element in an elite Ammonite name, the default should be that these lunar emblems represent this local deity. However, the iconography of Sîn of Harran used on the reliefs at Rujm al-Kursi suggests a more complicated situation. Was Sîn worshipped in Ammon alongside Yarah. ? Was Sîn's iconography applied to the West Semitic moon-god Yarah. ? Or were Sîn and Yarah. identified or otherwise thought to be the same deity? There is at present no way to answer the question definitively. Nonetheless, we do know that Sîn of Harran played a special role in Neo-Assyrian expansion and management of the western part of the empire. In part, this seems to be related to the location of Harran as a kind of gateway to Assyria's western periphery, but also because of the ease with which one moon-god could be identified with another (Holloway 1995, pp. 279–308). This reality favors the conclusion that some kind of identification took place between Sîn of Harran and the West Semitic Yarah. .

Along different lines, iconographic analysis of the representation of deities in the first millennium BCE ancient Near East shows significant overlap and mixing of the iconographic attributes of the various local versions of the weather god and the moon-god. A particular point of similarity is their common attribute animal, the bull (Novák 2002; Ornan 2001, pp. 19–26). The mixing of such attributes is visible on seals, and reliefs on stelae and orthostats. Closest to Ammon is the stele from Bethsaida (Figure 8) that combines weather god, bull, and lunar symbolism in one figure (Bernett

23 An unprovenanced seal that may be from the region of Ammon seems to incorporate the boat motif, the stylized tree, and the anthropomorphic deity sitting on a bench facing an ankh (Keel and Uehlinger 1998, pp. 307, illus. 305a; Avigad and Sass 1997, p. 446, no. 1177).

and Keel 1998; Ornan 2001). The apparent amalgamation or merging of weather god and moon-god iconographic features might well have taken place in Ammon given the connection between a lunar crescent and bovine figures on Tall al-Mazar 18 and Tall al-ގUmeiri 11 (Figure 6c,d). Finally, as Keel and Uehlinger have shown, the night time celestial powers in general—the stars, Venus, and the moon—were popular in the iconography of Iron IIC seals from the Levant, similar to the way in which the sun and its iconography had been popular in the preceding Iron IIB (Keel and Uehlinger 1998, pp. 287–323). In this broad iconographic context, it is possible to see a further amalgamation of deity attributes in which state gods such as Yahweh and Milkom, who had already been identified with ގEl, took on the additional imagery of the moon (Keel and Uehlinger 1998, pp. 306–16). Along these lines, Theuer has called attention to the seal of Milkomގor as highlighting the connection between Milkom and the moon. On the seal (Tall al-ގUmeiri 4; *CSAJ*, pp. 312–13), the central register displays a four-winged scarab beetle flanked by two poles or standards with a ball (probably a full moon) inside a downward facing moon crescent. In addition to the clear lunar aspects of the iconography, Theuer notes that the second part of Milkomގor's name, which means "light," is a known epithet of the moon-god (Theuer 2000, p. 414). While we cannot reach a definitive conclusion on the amalgamation or identification of ގEl, the weather god (which was usually the state god), and the moon-god in Ammon, the evidence is suggestive that some such process did take place. In addition, the imperial context of the evidence, with all of its pressures and opportunities for cultural exchange, was ripe for such processes.24

**Figure 8.** Stele from the gate area of Bethsaida from (Keel 1998, fig. 107). Note the bull head and horns that form a crescent moon.


**Author details**

Daniela Melo1 , Susana Rocha1 , Susana Coimbra2,3 and Alice Santos Silva1 \*

1 UCIBIO, REQUIMTE, Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal

2 UCIBIO, REQUIMTE, Porto, Portugal

3 CESPU, Institute of research and Advanced Training in Health Sciences and Technologies (IINFACTS), Gandra-Paredes, Portugal

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**5. Conclusions**

#### **Figure 4.**

*Viscosity profiles of rice samples collected during drying. OOC, ordinary operating conditions; PROG, program; IN, representative sample of the loading; OUT, representative sample of the unloading.*

temperatures). For MC below 15%, the grain should be dried at milder conditions (lower grain temperatures) to avoid an increase of the HRYR.

• Based on these results, a two-stage drying program was proposed to improve the drying efficiency (drying rate and HRYR). This program was tested in a

*Improving the Efficiency of Rice Drying: Impact of Operational Variables on the Drying… DOI: http://dx.doi.org/10.5772/intechopen.112970*

commercial cross-flow dryer using a South American rice variety with promising results. The HRYR was reduced and the drying rate increased, compared to the runs performed at the ordinary operating conditions of the industry. Additionally, the cooking properties were not affected.

• Implementing this drying program would allow to increase the reception capacity of the rice industries and reduce the percentage of rice kernels with lower added value (broken kernels), improving the productivity of the industrial sector.


**4.2 Temperature treatments**

*Temperature Treatments;* when dormancy is caused by embryo factor, the seed is incubated at a low temperature (0–5°C) on a substratum for 3 to 10 days, allowing it to reach its optimum temperature. Germination necessitates the presence of this ingredient. - Take mustard, for example (Brassica campestrits). Before germinating at the proper temperature, some seeds required a brief period of incubation (from a few

hours to one to five days) at 40 to 50°C. (When using this approach, make sure the seed has a moisture level of no more than 15%, for example, paddy) (Oryza sativa). Breaking hard-seed ness in legumes with hot water treatment is also an effective strategy. The seeds are steeped in water at a temperature of 80°C for 1–5 minutes in this approach before putting for germination (depending up on the type of seed) [29].

### **4.3 Light treatments**

*Light Treatments;* some seeds do not germinate in dark thus are providing with continuous or periodic exposure of light is essential for example Lettuce (*Lactuca sativa*) required red light (660 nm) or white light is essential for germination to occur [20].


*Article* **The Epidemiology, Cost, and Occupational Context of Spinal Injuries Sustained While 'Working for Income' in NSW: A Record-Linkage Study**

**Lisa N. Sharwood 1,2,3,\*, Holger Mueller 4, Rebecca Q. Ivers 4,5, Bharat Vaikuntam 1,2, Tim Driscoll 6 and James W. Middleton 1,2,7**

Received: 8 September 2018; Accepted: 23 September 2018; Published: 27 September 2018

**Abstract:** This study aimed to describe the epidemiological characteristics, the occupational context, and the cost of hospitalised work-related traumatic spinal injuries, across New South Wales, Australia. A record-linkage study of hospitalised cases of work-related spinal injury (ICD10-AM code U73.0 or workers compensation) was conducted. Study period 2013–2016. Eight hundred and twenty-four individuals sustained work-related spinal injuries; 86.2% of whom were males and had a mean age of 46.6 years. Falls led to 50% of the injuries; predominantly falls from building/structures, ladders or between levels. Falls occurred predominantly in the construction industry (78%). Transport crashes caused 31% of injuries and 24% in heavy vehicles. Half of all the transport injuries occurred 'off road'. The external cause was coded as 'non-specific work activity' in 44.5% of cases; missing in 11.5%. Acute care bed days numbered at 13,302; total cost \$19,500,000. High numbers of work-related spinal injuries occurred in the construction industry; particularly falling from a height. Off-road transport-related injuries were significant and likely unaddressed by 'on-road' prevention policies. Medical record documentation was insufficient in injury mechanism and context specificity. Workers in the construction industry or those using vehicles off-road were at high risk of spinal injury, suggesting inefficient systems approaches or ineffective prevention policies. Reducing the use of non-specific external cause codes in patients' medical records would improve the measurement of policy effectiveness.

**Keywords:** workplace injuries; spinal trauma; record-linkage data


*2.9. Cytology*

The physician-collected ThinPrep PreservCyt cervical specimens were processed in the ThinPrep 2000 machine (Cytyc Corporation, Boxborough, Mass). All liquid-based cytology slides were stained according to the Pap method and all cytologic diagnosis were performed by trained specialist biotechnicians, following automated Focal Point evaluation of all slides. In case of abnormal cytology, a dedicated pathologist from the Cytology Unit of IEO reviewed cytology slides to confirm diagnosis. Results were reported according to the 2001 Bethesda Reporting System.


*Article* **Cloning and Partial Characterization of an Endo-**α**-(1**→**6)-**d**-Mannanase Gene from** *Bacillus circulans*

**Shiva kumar Angala 1,\*,**†**, Wei Li 1,**†**, Zuzana Palˇceková 1, Lu Zou 2, Todd L. Lowary 2, Michael R. McNeil 1 and Mary Jackson 1,\***

Received: 22 November 2019; Accepted: 6 December 2019; Published: 11 December 2019

**Abstract:** Mycobacteria produce two major lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), whose broad array of biological activities are tightly related to the fine details of their structure. However, the heterogeneity of these molecules in terms of internal and terminal covalent modifications and complex internal branching patterns represent significant obstacles to their structural characterization. Previously, an endo-α-(1→6)-D-mannanase from *Bacillus circulans* proved useful in cleaving the mannan backbone of LM and LAM, allowing the reducing end of these molecules to be identified as Man*p*-(1→6) [Man*p*-(1→2)]-Ino. Although first reported 45 years ago, no easily accessible form of this enzyme was available to the research community, a fact that may in part be explained by a lack of knowledge of its complete gene sequence. Here, we report on the successful cloning of the complete endo-α-(1→6)-D-mannanase gene from *Bacillus circulans* TN-31, herein referred to as *emn*. We further report on the successful production and purification of the glycosyl hydrolase domain of this enzyme and its use to gain further insight into its substrate specificity using synthetic mannoside acceptors as well as LM and phosphatidyl-*myo*-inositol mannoside precursors purified from mycobacteria.

**Keywords:** endo-α-(1→6)-d-mannase; mannoside; *Mycobacterium*; lipomannan; lipoarabinomannan; phosphatidylinositol mannosides