The effect of Mycobacterium tuberculosis CRISPR-associated Cas2 (Rv2816c) on stress response genes expression,morphology and macrophage survival of Mycobacterium smegmatis

Clustered regularly interspaced short palindromic repeats (CRISPR) are present in the genome of 40% bacteria and 90% archaea. CRISPR and accompanying Cas proteins constitute an adaptive immune system against disruptive mobile genetic elements. Two CRISPRs and 9 genes encoding CRISPR-associated proteins have been found in the genome of Mycobacterium tuberculosis. The CRISPR-associated Cas2 is an endoribonuclease required for the acquisition of new spacers. In this study, Cas2 encoded by Rv2816c was expressed in Mycobacterium smegmatis lacking CRISPR-Cas system and its role in stress responses of M. smegmatis in vitro and within macrophages was studied. We found that Cas2 mediated M. smegmatis stress response changes were associated with the altered expression of sigma factors which involved in mycobacterial stress response and virulence. We also found that Cas2 decreased the survival of M. smegmatis within macrophages. This study provides new insights on the role of Cas2.     

Polymorphism of Type I–F CRISPR/Cas system in Escherichia coli of phylogenetic group B2 and its application in genotyping

E. coli of phylogenetic group B2 is responsible for many extraintestinal infections, posing a great threat to health. The relatively polymorphic nature of CRISPR in phylogenetically related E. coli strains makes them potential markers for bacterial typing and evolutionary studies. In the current work, we investigated the occurrence and diversity of CRISPR/Cas system and explored its potential for genotyping. Type I–F CRISPR/Cas systems were found in 413 of 1190 strains of E. coli and exhibited the clustering within certain CCs and STs. And CRISPR spacer contents correlated well with MLST types. The divergence analysis of CRISPR showed stronger discriminatory power than MLST, and CRISPR polymorphism was instrumental for differentiating highly closely related strains. The timeline of spacer acquisition and deletion provided important information for inferring the evolution model between distinct serotypes. Identical spacer sequences were shared by strains with the same H-antigen type but not strains with the same O-antigen type. The homology between spacers and antibiotic-resistant plasmids demonstrated the role of Type I–F system in limiting the acquisition of antimicrobial resistance. Collectively, our data presents the dynamic nature of Type I–F CRISPR in E. coli of phylogenetic group B2 and provides new insights into the application of CRISPR-based typing in the species.     

CRISPR1上游侧翼序列用于大肠埃希菌和志贺菌鉴定的效果评价

目的 探讨基于成簇规律间隔的短回文重复序列（CRISPR）1上游侧翼序列对大肠埃希菌和志贺菌鉴定和评价效果。方法 通过BLAST重复序列识别并获得全基因组测序大肠埃希菌和志贺菌的CRISPRs和CRISPR相关基因（CRISPR-associated,cas）,并分析其种系;选取CRISPRs上、下游各500 bp侧翼序列,使用Clustal X进行序列比对;采用PCR方法扩增CRISPR1上游侧翼序列,以确定其对大肠埃希菌和志贺菌鉴定和评价效果。结果 73.4%（149/203）的大肠埃希菌存在I-E型CRISPR/Cas系统,包含了A、B1、D种系;8.4%（17/203）的大肠埃希菌存在I-F型CRISPR/Cas、17.2%（35/203）的大肠埃希菌不存在CRISPR/Cas,这2种大肠埃希菌均属于B2种系;9株志贺菌均存在I-E型CRISPR/Cas。在大肠埃希菌（B2种系外）、志贺菌CRISPR1上游和大肠埃希菌（B2种系）各存在61 bp侧翼序列,序列一致性为99%,且有种属特异性,PCR扩增此区域鉴定大肠埃希菌和志贺菌的灵敏度和特异度均>91%。结论 基于CRISPR1上游序列可用来鉴定大肠埃希菌和志贺菌,且具有很好的效果。     

A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes

Propionibacterium acnes plays a central role in the pathogenesis of acne and is responsible for severe opportunistic infections. Numerous typing schemes have been developed that allow the identification of phylotypes, but they are often insufficient to differentiate subtypes. To better understand the genetic diversity of this species and to perform epidemiological analyses, high throughput discriminant genotyping techniques are needed. Here we describe the development of a multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) method. Thirteen VNTRs were identified in the genome of P. acnes and were used to genotype a collection of clinical isolates. In addition, publically available sequencing data for 102 genomes were analyzed in silico, providing an MLVA genotype. The clustering of MLVA data was in perfect congruence with whole genome based clustering. Analysis of the clustered regularly interspaced short palindromic repeat (CRISPR) element uncovered new spacers, a supplementary source of genotypic information. The present MLVA₁₃ scheme and associated internet database represents a first line genotyping assay to investigate large number of isolates. Particular strains may then be submitted to full genome sequencing in order to better analyze their pathogenic potential.     

4株志贺菌无抗生素压力下连续传代90次的耐药表型及CRISPR/Cas系统变化

目的 分析无抗生素压力下连续传代90次的4株志贺菌耐药表型及成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）基因变化。方法 对临床分离的4株耐药谱不同的志贺菌进行无抗生素压力连续传代90次,传代结束后用琼脂稀释法检测传代前、后志贺菌最小抑菌浓度;用PCR对CRISPR位点进行扩增并测序,CRISPR Finder和Clustal X 2.1分析CRISPR位点的变化。结果 经无抗生素压力传代90次后,4株志贺菌对某些抗生素的敏感性有不同程度的增加：mel-sf1998024/zz对氨苄西林、头孢氨苄、头孢噻肟、氯霉素的耐药性降低,mel-s2014026/sx对诺氟沙星、甲氧苄啶的耐药性降低,mel-sf2004004/sx对氨苄西林、头孢呋辛、头孢噻肟、氯霉素、甲氧苄啶的耐药性降低,mel-sf2013004/bj对氯霉素的耐药性降低;志贺菌mel-sf1998024/zz和mel-sf2013004/bj经无抗生素压力传代后,CRISPR3位点3''的重复-间隔序列丢失,其中间隔序列匹配基因的编码产物是Cas蛋白。结论 志贺菌在无抗生素压力下,可降低或丢失对某些抗生素的耐药性。部分志贺菌CRISPR3位点的结构发生了变化,CRISPR3位点与cas基因可能存在共进化。     

Uropathogenic Escherichia coli are less likely than paired fecal E. coli to have CRISPR loci

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are short fragments of DNA that act as an adaptive immune system protecting bacteria against invasion by phages, plasmids or other forms of foreign DNA. Bacteria without a CRISPR locus may more readily adapt to environmental changes by acquiring foreign genetic material. Uropathogenic Escherichia coli (UPEC) live in a number of environments suggesting an ability to rapidly adapt to new environments. If UPEC are more adaptive than commensal E. coli we would expect that UPEC would have fewer CRISPR loci, and – if loci are present – that they would harbor fewer spacers than CRISPR loci in fecal E. coli. We tested this in vivo by comparing the number of CRISPR loci and spacers, and sensitivity to antibiotics (resistance is often obtained via plasmids) among 81 pairs of UPEC and fecal E. coli isolated from women with urinary tract infection. Each pair included one uropathogen and one commensal (fecal) sample from the same female patient.Fecal isolates had more repeats (p = 0.009) and more unique spacers (p < 0.0001) at four CRISPR loci than uropathogens. By contrast, uropathogens were more likely than fecal E. coli to be resistant to ampicillin, cefazolin and trimethoprim/sulfamethoxazole. However, no consistent association between CRISPRs and antibiotic resistance was identified. To our knowledge, this is the first study to compare fecal E. coli and pathogenic E. coli from the same individuals, and to test the association of CRISPR loci with antibiotic resistance. Our results suggest that the absence of CRISPR loci may make UPEC more susceptible to infection by phages or plasmids and allow them to adapt more quickly to various environments.     

肺炎克雷伯菌中CRISPR调控耐药机制及其分布特征与分离地点的关系

  目的  了解呼吸道标本肺炎克雷伯菌中成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats,CRISPR）对耐药的调控机制,并分析其CRISPR分布特征与分离地点的关系。  方法  收集并提取120株肺炎克雷伯菌的基因组DNA,通过扩增CRISPR/Cas（clustered regularly interspaced short palindromic repeats/CRISPR-associated）系统相关基因CRISPR 1、CRISPR 2来确定CRISPR阳性菌株。CRISPR阳性菌株的耐药表型用BD Phoenix-100细菌鉴定仪进行检测,利用CRISPR Target寻找间隔序列同源噬菌体或质粒,并在Center for Genomic Epidemiology上查找同源质粒或噬菌体的耐药信息并检测间隔序列所在菌株的耐药基因,分析两者携带耐药基因的关系。采用CRISPR Finder分析CRISPR并运用多序列比对分析间隔序列的一致性。  结果  CRISPR1、CRISPR2阳性率分别为12.50%和13.33%;间隔序列同源质粒与其所在菌株均携带共同的耐药基因,且菌株的耐药表型与其携带的耐药基因高度符合;相同地点菌株的CRISPR分布具有极高相似性。  结论  肺炎克雷伯菌通过将外来质粒的耐药基因片段整合到菌株的基因组中实现对菌株耐药性的调控;CRISPR中间隔序列的分布与菌株分离地点密切相关,为临床治疗和感染控制工作提供理论依据。     

O26:H11及NM大肠埃希菌CRISPR的分子分布特征及其与stx噬菌体的关系

目的 探讨O26：H11及NM血清型大肠埃希菌中成簇规律间隔短回文重复序列（CRISPR）的分子分布特征及其与stx噬菌体的关系。方法 135株O26：H11及NM血清型大肠埃希菌从NCBI数据库获取,利用CRT软件及CRISPR Finder提取CRISPR信息,并用Excel软件对间隔序列进行编号及分析CRISPR亚型,并分析CRISPR与stx噬菌体之间的关系。结果 135株O26：H11及NM血清型大肠埃希菌中均存在CRISPR结构,CRISPR1包括19个亚型,CRISPR 2.1包括22个亚型,CRISPR2.2包括1个亚型,CRISPR3-4包括1个亚型。stx噬菌体在CRISPR群组C中出现,stx⁺ 菌株比stx^-菌株拥有更多的间隔序列。结论 CRISPR位点在O26：H11或NM血清型大肠埃希菌中广泛存在,且存在着不同的亚型,stx噬菌体与CRISPR的分子分布特征有关,可能作为鉴定高毒菌株的分子靶标。     

Comparative genomic and phylogenetic analysis of a toxigenic clinical isolate of Corynebacterium diphtheriae strain B-D-16-78 from Malaysia

In this study, we report the comparative genomics and phylogenetic analysis of Corynebacterium diphtheriae strain B-D-16-78 that was isolated from a clinical specimen in 2016. The complete genome of C. diphtheriae strain B-D-16-78 was sequenced using PacBio Single Molecule, Real-Time sequencing technology and consists of a 2,474,151-bp circular chromosome with an average GC content of 53.56%. The core genome of C. diphtheriae was also deduced from a total of 74 strains with complete or draft genome sequences and the core genome-based phylogenetic analysis revealed close genetic relationship among strains that shared the same MLST allelic profile. In the context of CRISPR-Cas system, which confers adaptive immunity against re-invading DNA, 73 out of 86 spacer sequences were found to be unique to Malaysian strains which harboured only type-II-C and/or type-I-E-a systems. A total of 48 tox genes which code for the diphtheria toxin were retrieved from the 74 genomes and with the exception of one truncated gene, only nucleotide substitutions were detected when compared to the tox gene sequence of PW8. More than half were synonymous substitution and only two were nonsynonymous substitutions whereby H24Y was predicted to have a damaging effect on the protein function whilst T262V was predicted to be tolerated. Both toxigenic and non-toxigenic toxin-gene bearing strains have been isolated in Malaysia but the repeated isolation of toxigenic strains with the same MLST profile suggests the possibility of some of these strains may be circulating in the population. Hence, efforts to increase herd immunity should be continued and supported by an effective monitoring and surveillance system to track, manage and control outbreak of cases.     

CRISPR/Cas9基因编辑技术对抗病毒潜伏感染的应用进展

目前抗病毒治疗主要靶向产毒阶段的病毒蛋白酶系统,而对于具有潜伏感染特征的病毒无效,因此能够靶向病毒基因组的抗病毒药物将是未来对抗病毒潜伏感染的研究策略.CRISPR/Cas9作为一种近年快速发展的基因编辑技术,在对抗病毒潜伏感染方面取得一定的成果,如HIV、人致病疱疹病毒、HBV等.此文对其应用进展进行综述,并总结了逃逸突变体的产生、脱靶效应、载体安全性等问题,以期为研制有效的抗潜伏感染病毒的治疗方法提供参考依据.     

Prevalence of 5′ insertion mutants and analysis of single nucleotide polymorphism in the erythrocyte binding-like 1 (ebl-1) gene in Kenyan Plasmodium falciparum field isolates

Plasmodium merozoites attach to and invade red blood cells (RBCs) during the erythrocytic cycle. The invasion process requires recognition of RBC surface receptors by proteins of the Plasmodium Duffy binding like erythrocyte binding like (DBL-EBP) family. Clones and isolates of Plasmodium falciparum have varying abilities to utilize different RBC receptors, and multiple distinct pathways so far identified depend on glycophorins A, B, C, and as yet unidentified receptors. At present, five members of the DBL-EBP family have been identified in the P. falciparum genome, based on gene structure and amino acid sequence homology. The cardinal features of this family consist of conserved 5′ and 3′ cysteine-rich regions (regions II and VI, respectively) whose cysteine residues are highly conserved along with the majority of aromatic amino acids. In contrast to the single DBL-EBP family member in Plasmodium vivax, in P. falciparum all DBL-EBP family members have a duplication of the conserved 5′ cysteine-rich region denoted as the F1 and F2 domains. These cysteine-rich regions are considered crucial in recognition of erythrocyte receptors and it has been shown that several bind to glycophorins on the erythrocyte surface. Several studies, on both field isolates and laboratory strains have uncovered a relatively high degree of sequence polymorphism in the DBP-EBL genes. This study is now extended to include field isolates collected from sites within Kenya. DNA isolated from blood samples of infected patients was utilized to amplify the region I sequence of ebl-1 gene in order to investigate polymorphism in the region immediately adjacent to the 5′ cysteine-rich domains, and to determine the prevalence of an insertion mutant that effectively knocks out the gene.     

Comparative genome analysis of fish pathogen Flavobacterium columnare reveals extensive sequence diversity within the species

Flavobacterium columnare is one of the deadliest fish pathogens causing devastating mortality in various freshwater fish species globally. To gain an insight into bacterial genomic contents and structures, comparative genome analyses were performed using the reference and newly sequenced genomes of F. columnare including genomovar I, II and I/II strains isolated from Thailand, Europe and the USA. Bacterial genomes varied in size from 3.09 to 3.39 Mb (2714 to 3101 CDSs). The pan-genome analysis revealed open pan-genome nature of F. columnare strains, which possessed at least 4953 genes and tended to increase progressively with the addition of a new genome. Genomic islands (GIs) present in bacterial genomes were diverse, in which 65% (39 out of 60) of possible GIs were strain-specific. A CRISPR/cas investigation indicated at least two different CRISPR systems with varied spacer profiles. On the other hand, putative virulence genes, including those related to gliding motility, type IX secretion system (T9SS), outer membrane proteins (Omp), were equally distributed among F. columnare strains. The MLSA scheme categorized bacterial strains into nine different sequence types (ST 9–17). Phylogenetic analyses based on either 16S rRNA, MLSA and concatenated SNPs of core genome revealed the diversity of F. columnare strains. DNA homology analysis indicated that the estimated digital DNA-DNA hybridization (dDDH) between strains of genomovar I and II can be as low as 42.6%, while the three uniquely tilapia-originated strains from Thailand (1214, NK01 and 1215) were clearly dissimilar to other F. columnare strains as the dDDH values were only 27.7–30.4%. Collectively, this extensive diversity among bacterial strains suggested that species designation of F. columnare would potentially require re-emendation.     

Comparison of Internal Transcribed Spacers and Intergenic Spacer Regions of Five Common Iranian Sheep Bursate Nematodes

Background

Accurate identification of sheep nematodes is a critical point in epidemiological studies and monitoring of drug resistance in flocks. However, due to a close morphological similarity between the eggs and larval stages of many of these nematodes, such identification is not a trivial task. There are a number of studies showing that molecular targets in ribosomal DNA (Internal transcribed spacer 1, 2 and Intergenic spacer) are suitable for accurate identification of sheep bursate nematodes. The objective of present study was to compare the ITS1, ITS2 and IGS regions of Iranian common bursate nematodes in order to choose best target for specific identification methods.

Methods

The first and second internal transcribed spacers (ITS1and ITS2) and intergenic spacer (IGS) of the ribosomal DNA (rDNA) of 5 common Iranian bursate nematodes of sheep were sequenced. The sequences of some non–Iranian isolates were used for comparison in order to evaluate the variation in sequence homology between geographically different nematode populations.

Results

Comparison of the ITS1 and ITS2 sequences of Iranian nematodes showed greatest similarity among Teladorsagia circumcincta and Marshallagia marshalli of 94% and 88%, respectively. While Trichostrongylus colubriformis and M. marshalli showed the highest homology (99%) in the IGS sequences. Comparison of the spacer sequences of Iranian with non-Iranian isolates showed significantly higher variation in Haemonchus contortus compared to the other species.

Conclusion

Both the ITS1 and ITS2 sequences are convenient targets to have species-specific identification of Iranian bursate nematodes. On the other hand the IGS region may be a less suitable molecular target.     

CRISPR-based subtyping to track the evolutionary history of a global clone of Acinetobacter baumannii

Acinetobacter baumannii global clone 1 (GC1) is the second most common clone in the global population of A. baumannii isolates and a key cause of hospital-acquired infections. In this study, comparative analysis of the clustered regularly interspaced short palindromic repeats (CRISPR)-based sequence types (CST) was performed to determine the genetic relatedness and track patterns of descent among 187 GC1 isolates, as a complement to the evolutionary inferences from their multilocus sequence types and genome-wide single nucleotide polymorphism (SNP)-based phylogeny.The CST2 cluster, CST2 and all the CSTs descending from CST2, corresponded to GC1 lineage 1. This cluster included 143 of the 187 isolates showing a prevalent geographical distribution worldwide. A well-demarcated group of 13 CSTs, accounting for 33 of the 187 isolates, corresponded to GC1 lineage 2. All the CSTs of this group were characterized by the absence of spacer Ab-18. Many of the GC1 lineage 2 isolates had an epidemiological link to the Middle East and/or were obtained in military healthcare facilities. GC1 lineage 3 was a novel lineage that has so far been limited to Afghanistan, Pakistan and India. Diversification of A. baumannii GC1 into lineages and clades has probably been related to a dynamic expansion after passing a migration bottleneck to enter the hospital environment.We conclude that CRISPR-based subtyping is a convenient method to trace the evolutionary history of particular bacterial clones, such as A. baumannii GC1.     

Distribution and characteristics of SGI1/PGI2 genomic island from Proteus strains in China

The emergence of multidrug-resistant Salmonella genomic island 1 (SGI1) and Proteus genomic island (PGI) bearing P. mirabilis present a serious threat to public health. In this study, we screened 288 Proteus isolates recovered from seven provinces in China. Fourteen strains (4.9%) all belonged to P. mirabilis were positive for SGI1/PGI2, including twelve from clinical samples (5.3%) and two from food (3.3%). A Blastn search against GenBank and phylogenetic analyses identified eight different SGI1 variants and one PGI2 variant from the fourteen SGI1/PGI2 variants. All SGI1 variants shared a common backbone and harbored different resistance gene(s), except the sul1 gene at its multidrug-resistant (MDR) region. Among the variants, three novel SGI1 variants, designated as SGI1-PmCA11, SGI1-PmCA14 and SGI1-PmCA46, contained different gene cassettes, which were similar to sequences in plasmids or class 1 integrons of Klebsiella pneumoniae, P. mirabilis, Escherichia coli and Salmonella. Moreover, one novel PGI2, designated as PGI2-PmCA72, had an identical gene cassette to the first class 1 integron from PGI2 (GenBank accession no. MG201402.1) in P. mirabilis, but varied due to missing, replaced, inserted and inverted gene clusters. The four novel SGI1/PGI2 variants contained the cmlA5, dfrA14, bla_OXA-10, aadA15, bla_OXA-1, catB3 and dfrA16 resistance genes, which have never been reported in SGI1/PGI2 variants. Phenotypically, all fourteen SGI1/PGI2-containing strains showed multidrug resistance. All except four strains were resistant to the first, or the second and/or-third generation cephalosporins. Considering the increasing number and the emergence of new SGI1/PGI2 variants, further surveillance is needed to prevent the spreading of the MDR genomic islands among Proteus isolates from human and food.     

Genetic diversity of clinical Salmonella enterica serovar Typhimurium in a university hospital of south Tunisia, 2000–2013

Typhimurium is one of the main Salmonella serovar responsible for non-typhoidal gastro-enteritis in Tunisia. Here, we aimed to assess the genetic diversity of 88 clinical Salmonella Typhimurium strains recovered during 14 years from 2000 to 2013. Phage typing, CRISPR polymorphisms (CRISPOL), pulsed-field gel electrophoresis (PFGE), multi-locus variable-number tandem repeat analysis (MLVA) and Whole genome sequencing (WGS) were used to study the relatedness and spatio-temporal evolution of Salmonella Typhimurium populations (Typhimurium (n = 81), monophasic (n = 3) and nonmotile (n = 4) variants).Seven-locus MLST from whole genome assemblies showed that all isolates, except one, belonged to ST19. The isolates were divided into 10 definitive phage (DT) types, dominated by DT104-L (39.8%), DT41 (14.8%), DT116 (11.4%) and DT120 (5.7%). Fifty-seven MLVA patterns (DI, 0.978) were obtained compared to 11 different CRISPOL types and 15 PFGE types (DI,0.845). For cgMLST analysis, 20 profiles were found. A total of 3056 SNPs were identified from the whole genome of the 88 Salmonella Typhimurium isolates. These SNPs resolved these isolates into 86 SNP haplotypes. The phylogeny result allocated most Salmonella Typhimurium isolates into four distinct clades and seven subclades. Genetic diversity between the four clades ranged in the order of 249 to 720 nucleotide changes. The prevalent phage type DT104L formed a major clade on the phylogenetic tree. Pairwise SNP differences between the strains of this clade ranged between 0 and 59.SNP-based WGS typing seems to be the most valuable molecular markers for studying the evolutionary relationships of homogeneous serovar Typhimurium isolates.     

Characterization of the Lancefield group C streptococcus 16S-23S RNA gene intergenic spacer and its potential for identification and sub-specific typing.

The 16S-23S RNA gene intergenic spacers of isolates of Streptococcus equi (n = 5), S. zooepidemicus (n = 5), S. equisimilis (n = 3) and S. dysgalactiae (n = 2) were sequenced and compared. There were distinct regions within the spacer, arranged in the order 1-9 for all S. equi and one S. zooepidemicus isolate and 1,2 and 4-9 for the remaining isolates. Region 4 was identical to the tRNA(ala) gene found in the 16S-23S intergenic spacers of other streptococci. Regions 1, 5, 6 and 7 had distinct variations, each conserved in different isolates. However, amongst the intergenic spacers there were different combinations of variant regions, suggesting a role for DNA recombination in their evolution. The intergenic spacer of all isolates of S. equi and one S. zooepidemicus isolate were almost identical. Primers derived from the variant sequences of regions 1 and 5 to 6 were used to group all S. zooepidemicus (n = 17) and S. equi (n = 5) into 1 of 8 types by polymerase chain reaction; three S. zooepidemicus isolates typed the same as S. equi. S. equi and S. zooepidemicus were clearly distinguishable from S. equisimilis and S. dysgalactiae which had shorter regions 5 and 6 and no region 7. Most homology for the group C sequences was found in previously published sequences for the 16S-23S intergenic spacers of S. anginosis, S. constellatus, S. intermedius, S. salivarius and S. agalactiae. A 75-90 nucleotide length shared with S. anginosus and S. intermedius in opposite orientations in the two main variants of region 6 supported the role for DNA recombination in the evolution of the spacer. The 16S-23S intergenic spacers indicate that S. zooepidemicus was the archetypal species for S. equi and that both are genetically more distant from S. equisimilis and S. dysgalactiae. The intergenic spacer can be used to identify specifically the group C streptococci and as an epidemiological marker for S. zooepidemicus.     

Changes in antimicrobial resistance and molecular attributes of Salmonellae causing enteric fever in Kolkata,India, 2014–2018

Globally, enteric fever caused by Salmonella Typhi (S. Typhi, ST) and S. Paratyphi A (SPA) remain one of the major diseases of public health importance. In this study, a total of 457 (380 ST, 77 SPA) blood isolates were collected from three tertiary care hospitals in Kolkata during 2014–18. Additionally, 66 (3.4%) ST and 5 (0.25%) SPA were recovered from blood culture of 1962 patients attending OPD of one pediatric hospital during 2016–18. The study isolates were tested for antimicrobial resistance (AMR) profiles; AMR genes; molecular sub-types by PFGE, MLVA and CRISPR. Among the total 446 ST and 82 SPA isolates, fluoroquinolone (FQ) resistance was very common in both serovars. Ciprofloxacin resistance of 24.9% and 9.8% & ofloxacin resistance of 20.9% and 87.8% were found in ST and SPA respectively. Majority (>70%) of the isolates showed decreased susceptibility to ciprofloxacin (DCS). A single point mutation in gyrA gene (S83F) was responsible for causing DCS in 37.5% (n = 42/112) ST and 63% (n = 46/73) SPA isolates. Multidrug resistance (MDR) was found only in 3.4% ST isolates and encoded the genes bla_TEM-1, catA, sul, strA-strB, class 1 integron with dfrA7. All MDR ST (n = 15) possessed non-conjugative non-IncHI1 (180 kb) plasmid except one having conjugative IncHI1 (230 kb) plasmid and one without plasmid. The MDR genes were integrated near chromosomal cyaA gene site in ST with/without the presence of plasmid (nonIncH1). Almost 65.7% resistant ST belonged to H58 haplotype. PFGE showed clonally related isolates with 81% similarity in ST and 87% in SPA. Similarly, CRISPR typing showed less diversity among the isolates. However, the isolates (ST and SPA) were found to be more diverse by MLVA typing (D value 0.987 and 0.938). The study reports decrease in MDR and increase in FQ resistance among typhoidal Salmonella isolates over the years giving interesting information for enteric fever treatment.     

Redefining MDR-TB: Comparison of Mycobacterium tuberculosis clinical isolates from Russia and Taiwan

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are global challenges due to the limited number of effective drugs for treatment. Treatment with less than 4–5 effective drugs might lead to the further emergence of drug resistance and poor clinical outcomes. For better prediction of treatment outcomes, we compared drug-resistance profiles of consecutive clinical MDR Mycobacterium tuberculosis isolates from high- and low-burden settings.This was a retrospective cohort study. We analysed 225 and 229 MDR isolates from Moscow (Russia) and Taiwan, respectively, obtained between 2014 and 2015. Drug susceptibility testing was performed by the Bactec MGIT 960 automated system and the agar proportion method. Detection of resistance-associated mutations in the M. tuberculosis genome was carried out by an array and/or sequencing of selected loci.The principal differences between resistance profiles of MDR isolates in the two countries were the percentages of pre-XDR (40.9% vs. 14.8%) and XDR (34.7% vs. 1.7%) isolates, both of which were significantly higher in Moscow isolates. Forty-eight (33%) of 147 MDR and pre-XDR Russian isolates fall into a group with less than four effective drugs, which accounts for 40% (N = 120) of these isolates. The other 60% in this group were XDR strains (N = 72). Consequently, the average number of effective anti-tuberculosis drugs for MDR-TB treatment was lower for Russian isolates (3 vs. 7). Furthermore, a notable percentage (9%) of isolates resistant to kanamycin harboured mutations in the whiB7 locus, which was not detected by molecular tests targeting common mutations in the rrs and eis loci. We found that 98.2% and 45.9% of MDR isolates from Moscow and Taiwan, respectively, were resistant to streptomycin.Molecular tests for detecting resistance to drugs other than rifampicin, isoniazid, fluoroquinolones, and second-line injectable drugs are needed for individualized therapy. The conventional MDR treatment schemes most probably fail in these cases due to the limited number of effective drugs.     

Extended-Spectrum β-Lactamase– and AmpC-Producing Enterobacteria in Healthy Broiler Chickens,Germany

During 2010, we evaluated the presence of extended-spectrum β-lactamase– and AmpC-producing enterobacteria in broiler chickens at slaughter. Samples (70 carcasses and 51 ceca) from 4 flocks were analyzed by direct plating and after enrichment. Extended-spectrum β-lactamase producers were found in 88.6% and 72.5% of carcasses and ceca, respectively; AmpC producers were found in 52.9% and 56.9% of carcasses and ceca, respectively. Most isolates were identified as Escherichia coli; Enterobacter cloacae (cecum) and Proteus mirabilis (carcass) were found in 2 samples each. Molecular characterization revealed the domination of CTX-M genes; plasmidic AmpC was CIT-like. Phylogenetic grouping of E. coli showed types A (31.5%), B1 (20.2%), B2 (13.5%), and D (34.8%). These findings provide evidence that healthy broilers in Germany are a source for the dissemination of transmissible resistance mechanisms in enterobacteria brought from the rearing environment into the food chain during slaughtering.