Prophage Types Research Articles

Evolution and zoonotic risk of O1:K1 and O2:K1 avian pathogenic Escherichia coli.

The O1 and O2 serogroups of avian pathogenic E. coli (APEC) and human extraintestinal pathogenic E. coli (huExPEC) are closely related, but their evolutionary relationships need to be further elucidated. This study classified nineteen O1 and O2 APEC into rpoB sequence types (RSTs) and compared them with reference huExPEC using molecular prophage typing, virulence and antibiotic resistance gene profiling, and comparative genomics. Most O1:K1 and O2:K1 APEC (73.7%) were classified as RST46-1 and RST47-9. RST47-9 is unique to Korean O1 APEC and likely derives from RST46-1 APEC. The six APEC showed high genome coverage/identity with the Korean RST46-1 huExPEC. Based on RST network and comparative genomics, we hypothesized that the O1 antigen first appeared in RST19-1 and O2 in RST24-1 E. coli in humans. Then, O1 and O2-antigen horizontally transferred to human RST46-1, where a unique K1 capsule (K1-cps) first appeared. The Korean APEC and huExPEC share evolutionary CRISPR spacers but differ in molecular antibiograms and prophage contents. Thus, RST46-1 huExPEC transmitted and evolved in poultry. The zoonotic risks remain unknown, but the substantial virulence of the RST46-1 APEC indicates that the reverse zoonotic risk of huExPEC in poultry is alarming.

Potential habitat suitability of Candidatus Liberibacter asiaticus and genetic diversity of its prophages across China.

This study offers significant insights into the distribution and genetic diversity of three types of prophages associated with Candidatus Liberibacter asiaticus (CLas) in China. Our predictions underscore the implications of climate change on the future distribution of CLas. These findings contribute to a better understanding of Huanglongbing management strategies and can facilitate the development of effective measures to control the spread of this devastating disease within the citrus industry.

Revisiting typing systems for group B Streptococcus prophages: an application in prophage detection and classification in group B Streptococcus isolates from Argentina.

Group B Streptococcus (GBS) causes severe infections in neonates and adults with comorbidities. Prophages have been reported to contribute to GBS evolution and pathogenicity. However, no studies are available to date on the presence and diversity of prophages in GBS isolates from humans in South America. This study provides insights into the prophage content of 365 GBS isolates collected from clinical samples in the context of an Argentinean multicentric study. Using whole-genome sequence data, we implemented two previously proposed methods for prophage typing: a PCR approach (carried out in silico) coupled with a blastx-based method to classify prophages based on their prophage group and integrase type, respectively. We manually searched the genomes and identified 325 prophages. However, only 80% of prophages could be accurately categorized with the previous approaches. Integration of phylogenetic analysis, prophage group and integrase type allowed for all to be classified into 19 prophage types, which correlated with GBS clonal complex grouping. The revised prophage typing approach was additionally improved by using a blastn search after enriching the database with ten new genes for prophage group classification combined with the existing integrase typing method. This modified and integrated typing system was applied to the analysis of 615 GBS genomes (365 GBS from Argentina and 250 from public databases), which revealed 29 prophage types, including two novel integrase subtypes. Their characterization and comparative analysis revealed major differences in the lysogeny and replication modules. Genes related to bacterial fitness, virulence or adaptation to stressful environments were detected in all prophage types. Considering prophage prevalence, distribution and their association with bacterial virulence, it is important to study their role in GBS epidemiology. In this context, we propose the use of an improved and integrated prophage typing system suitable for rapid phage detection and classification with little computational processing.

Diversity and Complexity of CTXΦ and Pre-CTXΦ Families in Vibrio cholerae from Seventh Pandemic.

CTXΦ is a lysogenic filamentous phage that carries the genes encoding cholera toxin (ctxAB), the main virulence factor of Vibrio cholerae. The toxigenic conversion of environmental V. cholerae strains through CTXΦ lysogenic infection is crucial for the emergence of new pathogenic clones. A special allelic form of CTXΦ, called pre-CTXΦ, is a precursor of CTXΦ and without ctxAB. Different members of the pre-CTXΦ and CTXΦ families are distinguished by the sequence of the transcriptional repressor-coding gene rstR. Multiple rstR alleles can coexist within a single strain, demonstrating the diverse structure and complex genomic integration patterns of CTXΦ/pre-CTXΦ prophage on the chromosome. Exploration of the diversity and co-integration patterns of CTXΦ/pre-CTXΦ prophages in V. cholerae can help to understand the evolution of this phage family. In this study, 21 V. cholerae strains, which were shown to carry the CTXΦ/pre-CTXΦ prophages as opposed to typical CTXETΦ-RS1 structure, were selected from approximately 1000 strains with diverse genomes. We identified two CTXΦ members and six pre-CTXΦ members with distinct rstR alleles, revealing complex chromosomal DNA integration patterns and arrangements of different prophages in these strains. Promoter activity assays showed that the transcriptional repressor RstR protected against CTXΦ superinfection by preventing the replication and integration of CTXΦ/pre-CTXΦ phages containing the same rstR allele, supporting the co-integration of the diverse CTXΦ/pre-CTXΦ members observed. The numbers and types of prophages and their co-integration arrangements in serogroup O139 strains were more complex than those in serogroup O1 strains. Also, these CTXΦ/pre-CTXΦ members were shown to present the bloom period of the CTXΦ/pre-CTXΦ family during wave 2 of the seventh cholera pandemic. Together, these analyses deepen our comprehension of the genetic variation of CTXΦ and pre-CTXΦ and provide insights into the evolution of the CTXΦ/pre-CTXΦ family in the seventh cholera pandemic.

Whole genome sequences of 135 “Candidatus Liberibacter asiaticus” strains from China

“Candidatus Liberibacter asiaticus” (CLas) is a phloem-limited alpha-proteobacteria causing Citrus Huanglongbing, the destructive disease currently threatening global citrus industry. Genomic analyses of CLas provide insights into its evolution and biology. Here, we sequenced and assembled whole genomes of 135 CLas strains originally from 20 citrus cultivars collected at ten citrus-growing provinces in China. The resulting dataset comprised 135 CLas genomes ranging from 1,221,309 bp to 1,308,521 bp, with an average coverage of 675X. Prophage typing showed that 44 strains contained Type 1 prophage, 89 strains contained Type 2 prophage, 44 strains contained Type 3 prophage, and 34 of them contained more than one type of prophage/phage. The SNP calling identified a total of 5,090 SNPs. Genome-based phylogenetic analysis revealed two major clades among CLas strains, with Clade I dominated by CLas strains containing Type 1 prophage (79/95) and Clade II dominated by CLas strains containing Type 1 or Type 3 prophage (80/95). This CLas genome dataset provides valuable resources for studying genetic diversity and evolutionary pattern of CLas strains.

Comparative genomics reveals high genetic similarity among strains of Salmonella enterica serovar Infantis isolated from multiple sources in Brazil.

Salmonella enterica serovar Infantis (Salmonella Infantis) is a zoonotic, ubiquitous and foodborne pathogen of worldwide distribution. Despite Brazil's relevance as a major meat exporter, few studies were conducted to characterize strains of this serovar by genomic analyses in this country. Therefore, this study aimed to assess the diversity of 80 Salmonella Infantis strains isolated from veterinary, food and human sources in Brazil between 2013 and 2018 by comparative genomic analyses. Additional genomes of non-Brazilian countries (n=18) were included for comparison purposes in some analyses. Analyses of whole-genome multi-locus sequence typing (wgMLST), using PGAdb-builder, and of fragmented genomes, using Gegenees, were conducted to compare the 80 Brazilian strains to the 18 non-Brazilian genomes. Pangenome analyses and calculations were performed for all Salmonella Infantis genomes analyzed. The presence of prophages was determined using PHASTER for the 80 Brazilian strains. The genome plasticity using BLAST Ring Image Generator (BRIG) and gene synteny using Mauve were evaluated for 20 selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Unique orthologous protein clusters were searched in ten selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. wgMLST and Gegenees showed a high genomic similarity among some Brazilian Salmonella Infantis genomes, and also the correlation of some clusters with non-Brazilian genomes. Gegenees also showed an overall similarity >91% among all Salmonella Infantis genomes. Pangenome calculations revealed an open pangenome for all Salmonella Infantis subsets analyzed and a high gene content in the core genomes. Fifteen types of prophages were detected among 97.5% of the Brazilian strains. BRIG and Mauve demonstrated a high structural similarity among the Brazilian and non-Brazilian isolates. Unique orthologous protein clusters related to biological processes, molecular functions, and cellular components were detected among Brazilian and non-Brazilian genomes. The results presented using different genomic approaches emphasized the significant genomic similarity among Brazilian Salmonella Infantis genomes analyzed, suggesting wide distribution of closely related genotypes among diverse sources in Brazil. The data generated contributed to novel information regarding the genomic diversity of Brazilian and non-Brazilian Salmonella Infantis in comparison. The different genetically related subtypes of Salmonella Infantis from Brazil can either occur exclusively within the country, or also in other countries, suggesting that some exportation of the Brazilian genotypes may have already occurred.

Diversity of 'Candidatus Liberibacter asiaticus' Strains in Texas Revealed by Prophage Sequence Analyses.

Prophages/phages are important components of the genome of 'Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

Prophage Carriage and Genetic Diversity within Environmental Isolates of Clostridioides difficile.

Clostridioides difficile is an important human pathogen causing antibiotic-associated diarrhoea worldwide. Besides using antibiotics for treatment, the interest in bacteriophages as an alternative therapeutic option has increased. Prophage abundance and genetic diversity are well-documented in clinical strains, but the carriage of prophages in environmental strains of C. difficile has not yet been explored. Thus, the prevalence and genetic diversity of integrated prophages in the genomes of 166 environmental C. difficile isolates were identified. In addition, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems were determined in the genomes of prophage regions. Predicted prophages and CRISPR-Cas systems were identified by using the PHASTER web server and CRISPRCasFinder, respectively. Phylogenetic relationships among predicated prophages were also constructed based on phage-related genes, terminase large (TerL) subunits and LysM. Among 372 intact prophages, the predominant prophages were phiCDHM1, phiCDHM19, phiMMP01, phiCD506, phiCD27, phiCD211, phiMMP03, and phiC2, followed by phiMMP02, phiCDKM9, phiCD6356, phiCDKM15, and phiCD505. Two newly discovered siphoviruses, phiSM101- and phivB_CpeS-CP51-like Clostridium phages, were identified in two C. difficile genomes. Most prophages were found in sequence types (STs) ST11, ST3, ST8, ST109, and ST2, followed by ST6, ST17, ST4, ST5, ST44, and ST58. An obvious correlation was found between prophage types and STs/ribotypes. Most predicated prophages carry CRISPR arrays. Some prophages carry several gene products, such as accessory gene regulator (Agr), putative spore protease, and abortive infection (Abi) systems. This study shows that prophage carriage, along with genetic diversity and their CRISPR arrays, may play a role in the biology, lifestyle, and fitness of their host strains.

Molecular Characterization and Phage Typing of Methicillin-resistant Staphylococcus aureus Isolated from Clinical Samples in Isfahan, Iran

Background: In hospitals and communities, Methicillin-resistant Staphylococcus aureus (MRSA) plays a critical role due to its ability to acquire resistance against several antibiotics and play a role in the spread of diseases. Objectives: This research aimed to investigate the pattern of antibiotic resistance in MRSA isolates and perform molecular typing of MRSA isolates using various elements, including SCCmec type, ccr type, prophage type, and gene toxin profiles. Methods: The research spanned 20 months at Al-Zahra Hospital in Isfahan and involved 148 isolates from various anatomical sites. The isolates were evaluated for their antibiotic susceptibility patterns. They were characterized by screening for SCCmec typing, ccr typing, phage typing, and PCR profiling of pvl, hlb, sak, eta, and tst toxin genes. Results: From 148 total S. aureus isolates, 42% (n = 62) were methicillin-resistant. The MRSA isolates demonstrated substantial resistance to penicillin and ciprofloxacin, and 90.3% of MRSA isolates were multiple-drug resistant. Also, SCCmec types III, I, and IV were identified in 45.16%, 35.48%, and 19.35% of MRSA isolates, respectively. Also, seven prophage patterns and 15 toxin patterns were detected among MRSA isolates. Conclusions: Multi-drug resistance is common among MRSA isolates. The only effective drug among the investigated antibiotics was chloramphenicol. The MRSA isolates can be controlled by changing the prescribing procedure of antibiotics and applying infection control strategies. The studied MRSA isolates can cause a wide range of diseases due to having several bacteriophages that encode virulence factors. Identification of different types of prophages may be useful in predicting such pathogenic agents.

Characterization and Diversity of Klebsiella pneumoniae Prophages.

Klebsiella pneumoniae is a common human commensal and opportunistic pathogen. In recent years, the clinical isolation and resistance rates of K. pneumoniae have shown a yearly increase, leading to a special interest in mobile genetic elements. Prophages are a representative class of mobile genetic elements that can carry host-friendly genes, transfer horizontally between strains, and coevolve with the host's genome. In this study, we identified 15,946 prophages from the genomes of 1437 fully assembled K. pneumoniae deposited in the NCBI database, with 9755 prophages on chromosomes and 6191 prophages on plasmids. We found prophages to be notably diverse and widely disseminated in the K. pneumoniae genomes. The K. pneumoniae prophages encoded multiple putative virulence factors and antibiotic resistance genes. The comparison of strain types with prophage types suggests that the two may be related. The differences in GC content between the same type of prophages and the genomic region in which they were located indicates the alien properties of the prophages. The overall distribution of GC content suggests that prophages integrated on chromosomes and plasmids may have different evolutionary characteristics. These results suggest a high prevalence of prophages in the K. pneumoniae genome and highlight the effect of prophages on strain characterization.

Discovery of a novel filamentous prophage in the genome of the Mimosa pudica microsymbiont Cupriavidus taiwanensis STM 6018.

Integrated virus genomes (prophages) are commonly found in sequenced bacterial genomes but have rarely been described in detail for rhizobial genomes. Cupriavidus taiwanensis STM 6018 is a rhizobial Betaproteobacteria strain that was isolated in 2006 from a root nodule of a Mimosa pudica host in French Guiana, South America. Here we describe features of the genome of STM 6018, focusing on the characterization of two different types of prophages that have been identified in its genome. The draft genome of STM 6018 is 6,553,639 bp, and consists of 80 scaffolds, containing 5,864 protein-coding genes and 61 RNA genes. STM 6018 contains all the nodulation and nitrogen fixation gene clusters common to symbiotic Cupriavidus species; sharing >99.97% bp identity homology to the nod/nif/noeM gene clusters from C. taiwanensis LMG19424T and "Cupriavidus neocalidonicus" STM 6070. The STM 6018 genome contains the genomes of two prophages: one complete Mu-like capsular phage and one filamentous phage, which integrates into a putative dif site. This is the first characterization of a filamentous phage found within the genome of a rhizobial strain. Further examination of sequenced rhizobial genomes identified filamentous prophage sequences in several Beta-rhizobial strains but not in any Alphaproteobacterial rhizobia.

Genomic characterization of Salmonella enterica serovar Choleraesuis from Brazil reveals a swine gallbladder isolate harboring colistin resistance gene mcr-1.1.

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) is a swine-adapted serovar associated to invasive infections in humans. In Brazil, data of strains of this serovar are scarce. In the present study, six S. Choleraesuis strains of animal (n = 5) and human (n = 1) origin from Brazil were screened for phenotypic antimicrobial resistance using disk-diffusion assay and using whole-genome sequencing data to search for antimicrobial resistance genes, plasmids, prophages, and Salmonella pathogenicity islands (SPIs). Its genetic relatedness was evaluated by MLST and SNP analysis. A single isolate from swine gallbladder harbored the colistin resistance gene mcr-1.1 into a IncX4 plasmid. In the six strains analyzed, resistance was found to tetracycline, nalidixic acid, ciprofloxacin, ampicillin, piperacillin, streptomycin, cefazoline, gentamycin, sulfamethoxazole-trimethoprim, and choloramphenicol, along with resistance genes aac(6')-Iaa, aac(3)-IV, aph(3'')-Ib, aph(6)-Id, aph(4)-Ia, aadA1, aph(3')-IIa, blaTEM-1A, floR, sul1, sul2, tet(B), drfA1, erm(B), mph(B), lnu(G), qacE, and gyrA point mutation Serine83 → Tyrosine and parC Threonine57 → Serine. Furthermore, IncF and IncH plasmids, ten SPIs, and seven prophage types were detected. All strains were assigned to ST145 and five belonged to a common SNP cluster of S. Choleraesuis strains from Brazil. The presence of S. Choleraesuis isolated from animals harboring relevant antimicrobial resistance profiles and virulence determinants reinforced the urge for enhanced surveillance to avoid its transmission to humans through food items.

Global population structure of the Serratia marcescens complex and identification of hospital-adapted lineages in the complex.

Serratia marcescens is an important nosocomial pathogen causing various opportunistic infections, such as urinary tract infections, bacteremia and sometimes even hospital outbreaks. The recent emergence and spread of multidrug-resistant (MDR) strains further pose serious threats to global public health. This bacterium is also ubiquitously found in natural environments, but the genomic differences between clinical and environmental isolates are not clear, including those between S. marcescens and its close relatives. In this study, we performed a large-scale genome analysis of S. marcescens and closely related species (referred to as the ‘ S. marcescens complex’), including more than 200 clinical and environmental strains newly sequenced here. Our analysis revealed their phylogenetic relationships and complex global population structure, comprising 14 clades, which were defined based on whole-genome average nucleotide identity. Clades 10, 11, 12 and 13 corresponded to S. nematodiphila , S. marcescens sensu stricto, S. ureilytica and S. surfactantfaciens, respectively. Several clades exhibited distinct genome sizes and GC contents and a negative correlation of these genomic parameters was observed in each clade, which was associated with the acquisition of mobile genetic elements (MGEs), but different types of MGEs, plasmids or prophages (and other integrative elements), were found to contribute to the generation of these genomic variations. Importantly, clades 1 and 2 mostly comprised clinical or hospital environment isolates and accumulated a wide range of antimicrobial resistance genes, including various extended-spectrum β-lactamase and carbapenemase genes, and fluoroquinolone target site mutations, leading to a high proportion of MDR strains. This finding suggests that clades 1 and 2 represent hospital-adapted lineages in the S. marcescens complex although their potential virulence is currently unknown. These data provide an important genomic basis for reconsidering the classification of this group of bacteria and reveal novel insights into their evolution, biology and differential importance in clinical settings.

Assessment of the lysogenic status in the lactic acid bacterium O. oeni during the spontaneous malolactic fermentation of red wines

After alcoholic fermentation, most wines undergo malolactic fermentation (MLF), driven by the lactic acid bacterium Oenococcus oeni, which improves their organoleptic properties and microbiological stability. Prophages were recently shown to be notably diverse and widely disseminated in O. oeni genomes. Such in silico predictions confirmed previous cultivation-based approaches which showed frequent lysis of strains upon treatment with the inducing agent mitomycin C. Both strategies used to assess lysogeny in the species were so far applied to a number of strains collected from distinct countries, wineries, cepages and fermentation processes. Results may not therefore be representative of the lysogenic population in natural communities driving the MLF during winemaking. Here we report the prevalence of lysogeny during winemaking in three wineries in the Bordeaux area. The dominant LAB population was collected in 11 red wines upon completion of MLF. Using VNTR and prophage typing analyses, our data confirm the presence of lysogens in the population driving the spontaneous MLF in all tested wines, although lysogeny rates varied across wineries. Higher prevalence of lysogeny was associated to a reduced diversity in VNTR profiles, the dominance of a few prophage-types and presence of some bacterial genetic backgrounds that were particularly prone to lysogenization.

Bacteriomic Analyses of Asian Citrus Psyllid and Citrus Samples Infected With "Candidatus Liberibacter asiaticus" in Southern California and Huanglongbing Management Implications.

Citrus Huanglongbing (HLB; yellow shoot disease) is associated with an unculturable α-proteobacterium “Candidatus Liberibacter asiaticus” (CLas). HLB was found in southern California in 2012, and the current management strategy is based on suppression of the Asian citrus psyllid (Diaphorina citri) that transmits CLas and removal of confirmed CLas-positive trees. Little is known about Asian citrus psyllid-associated bacteria and citrus-associated bacteria in the HLB system. Such information is important in HLB management, particularly for accurate detection of CLas. Recent advancements in next-generation sequencing technology provide new opportunities to study HLB through genomic DNA sequence analyses (metagenomics). In this study, HLB-related bacteria in Asian citrus psyllid and citrus (represented by leaf midrib tissues) samples from southern California were analyzed. A metagenomic pipeline was developed to serve as a prototype for future bacteriomic research. This pipeline included steps of next-generation sequencing in Illumina platform, de novo assembly of Illumina reads, sequence classification using the Kaiju tool, acquisition of bacterial draft genome sequences, and taxonomic validation and diversity evaluation using average nucleotide identity. The identified bacteria in Asian citrus psyllids and citrus together included Bradyrhizobium, Buchnera, Burkholderia, “Candidatus Profftella armature,” “Candidatus Carsonella ruddii,” CLas, Mesorhizobium, Paraburkholderia, Pseudomonas, and Wolbachia. The whole genome of a CLas strain recently found in San Bernardino County was sequenced and classified into prophage typing group 1 (PTG-1), one of the five known CLas groups in California. Based on sequence similarity, Bradyrhizobium and Mesorhizobium were identified as possible source that could interfere with CLas detection using the 16S rRNA gene-based PCR commonly used for HLB diagnosis, particularly at low or zero CLas titer situation.

Genomic Analysis of Prophages Recovered from Listeria monocytogenes Lysogens Found in Seafood and Seafood-Related Environment.

A prophage is a phage-related sequence that is integrated into a bacterial chromosome. Prophages play an important role in bacterial evolution, survival, and persistence. To understand the impact of Listeria prophages on their host genome organizations, this work sequenced two L. monocytogenes strains (134LM and 036LM), previously identified as lysogens by mitomycin C induction. Draft genomes were generated with assembly sizes of 2,953,877 bp and 3,000,399 bp. One intact prophage (39,532 bp) was inserted into the comK gene of the 134LM genome. Two intact prophages (48,684 bp and 39,488 bp) were inserted in tRNA-Lys and elongation-factor genes of the 036LM genome. The findings confirmed the presence of three corresponding induced phages previously obtained by mitomycin C induction. Comparative genomic analysis of three prophages obtained in the newly sequenced lysogens with 61 prophages found in L. monocytogenes genomes, available in public databases, identified six major clusters using whole genome-based phylogenetic analysis. The results of the comparative genomic analysis of the prophage sequences provides knowledge about the diversity of Listeria prophages and their distribution among Listeria genomes in diverse environments, including different sources or geographical regions. In addition, the prophage sequences and their insertion sites contribute to the genomic diversity of L. monocytogenes genomes. These data of prophage sequences, prophage insertion sites, and prophage sequence comparisons, together with ANIb confirmation, could be useful for L. monocytogenes classification by prophages. One potential development could be refinement of prophage typing tools for monitoring or surveillance of L. monocytogenes contamination and transmission.

Genetic diversity and population structure of 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing in India based on the prophage types.

Huanglongbing (HLB), also known as 'citrus greening', is an extremely destructive disease of citrus worldwide. HLB is associated with three species of the fastidious proteobacterium, Candidatus Liberibacter asiaticus (CaLas), Ca. L. africanus and Ca. L. americanus with CaLas being the most widely distributed around the world and the only species detected and described so far in India, one of the major global citrus fruit producers. Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. Three types of prophages, Type 1, Type 2 and Type 3 have been identified and described in CaLas so far. In the present study, 441 CaLas isolates sampled across 18 Indian states were used for prophage typing. Based on detection of three prophage types by PCR, all the eight probable combinations of CaLas prophages were identified, including single Type 1 (26.5%), single Type 2 (18.8%), single Type 3 (1.4%), Type 1 + Type 2 (20.4%), Type 1 + Type 3 (12.5%), Type 2 + Type 3 (4.8%), Type 1 + Type 2 + Type 3 (11.3%) and None type (4.3%). Prophage types were confirmed by PCR amplicon sequencing and subsequent phylogenetic analysis. By discovery of all 3 prophages and based on genetic identity and genetic distance, CaLas populations from eighteen citrus growing states were separated into two major Prophage Typing Groups (PTGs): PTG1 and PTG2. The PTG1 comprised of CaLas from North-West India and PTG2 from rest of the country (North-East, Central and South India), and both major groups were further divided into two (PTG1-A, PTG1-B) and three (PTG2-A, PTG2-B and PTG2-C) subgroups respectively. The findings of CaLas population patterns provide evidence for independent origins of HLB-associated CaLas. CRISPR (clustered regularly interspaced short palindromic repeats) array was also detected in CaLas isolates. This is the first report evaluating the genetic variation of a large population of CaLas bacterium in India using the PCR markers from the prophage regions which would certainly assist the ongoing HLB management efforts in India.

Whole-Genome Sequencing Reveals the Presence of the blaCTX-M-65Gene in Extended-Spectrum β-Lactamase-Producing and Multi-Drug-Resistant Clones of Salmonella Serovar Infantis Isolated from Broiler Chicken Environments in the Galapagos Islands.

Salmonella Infantis, a common contaminant of poultry products, is known to harbor mobile genetic elements that confer multi-drug resistance (MDR) and have been detected in many continents. Here, we report four MDR S. Infantis strains recovered from poultry house environments in Santa Cruz Island of the Galapagos showing extended-spectrum β-lactamase (ESBL) resistance and reduced fluoroquinolone susceptibility. Whole-genome sequencing (WGS) revealed the presence of the ESBL-conferring blaCTX-M-65 gene in an IncFIB-like plasmid in three S. Infantis isolates. Multi-locus sequence typing (MLST) and single nucleotide variant/polymorphism (SNP) SNVPhyl analysis showed that the S. Infantis isolates belong to sequence type ST32, likely share a common ancestor, and are closely related (1–3 SNP difference) to blaCTX-M-65-containing clinical and veterinary S. Infantis isolates from the United States and Latin America. Furthermore, phylogenetic analysis of SNPs following core-genome alignment (i.e., ParSNP) inferred close relatedness between the S. Infantis isolates from Galapagos and the United States. Prophage typing confirmed the close relationship among the Galapagos S. Infantis and was useful in distinguishing them from the United States isolates. This is the first report of MDR blaCTX-M-65-containing S. Infantis in the Galapagos Islands and highlights the need for increased monitoring and surveillance programs to determine prevalence, sources, and reservoirs of MDR pathogens.

Characterization of methicillin-resistant Staphylococcus aureus strains in sewage treatment plants in Tehran, Iran.

Multidrug-resistant Staphylococcus aureus strains have been commonly found in hospitals and communities causing wide ranges of infections among humans and animals. Typing of these strains is a key factor to reveal their clonal dissemination in different regions. We investigated the prevalence and dissemination of different clonal groups of S. aureus with resistance phenotype to multiple antibiotics in two sewage treatment plants (STPs) in Tehran, Iran over four sampling occasions. A total of 576 S. aureus were isolated from the inlet, sludge and outlet. Of these, 80 were identified as methicillin-resistant S. aureus (MRSA) and were further characterized using a combination of Phene Plate (PhP) typing, staphylococcal cassette chromosome mec (SCCmec), ccr types, prophage and antibiotic-resistant profiling. In all, eight common type (CT) and 13 single PhP type were identified in both STPs, with one major CT accounting for 38.8% of the MRSA strains. These strains belonged to three prophage patterns and five prophage types with SCCmec type III being the predominant type. Resistance to 11 out of the 17 antibiotics tested was significantly (P < 0.0059) higher among the MRSA isolates than methicillin-sensitive S. aureus (MSSA) strains. The persistence of the strains in samples collected from the outlet of both STPs was 31.9% for MRSA and 23.1% for MSSA. These data indicated that while the sewage treatment process, in general, is still useful for removing most MRSA populations, some strains with SCCmec type III may have a better ability to survive the STP process.

Prophage Typing of Staphylococcus aureus Strains Isolated from Broiler Poultry.

Staphylococcus aureus is a well-known commensal and pathogen agent of many wild and domestic animals. A wide variety of infections can be caused by S. aureus, from suppurative skin infections to life-threatening septicemia. This study was conducted to determine the prophage typing and the pattern of antibiotic resistance of S. aureus isolated from broiler poultry before they have been slaughtered. In this study, 200 nasal and cloacal swab samples from 20 different flocks were collected for bacterial isolation. Staphylococci were identified using biochemical and molecular methods before being examined for mecA gene detections in all samples resistant to oxacillin and cefotaxime. The highest value of antibiotic resistance was observed against ciprofloxacin (94%), and the maximum value of susceptibility was to gentamicin (85%). Twenty-eight (27%) samples were resistant to oxacillin. In methicillin-resistance Staphylococcus aureus (MRSA) isolates, 5 prophage types were observed, where the SGB prophage with a frequency of 75% was identified as a dominant prophage; in isolates of S. aureus susceptible to methicillin, 8 prophage types were observed, where SGFa prophage with a frequency about 82% was the dominant prophage. The high prevalence of MRSA isolates can indicate the risk of transmission of these bacteria to the food cycle. Furthermore, existence of various prophages in these isolates can be considered a threat to public health in producing pathogenicity factors in this bacterium while also empowering other bacterial pathogenicity, even other bacterial genera.