Effect of Subinhibitory Concentrations of Antibiotics and Disinfectants on ISAba-Mediated Inactivation of Lipooligosaccharide Biosynthesis Genes in Acinetobacter baumannii

Inactivation of the lipooligosaccharide (LOS) biosynthesis genes lpxA, lpxC and lpxD by ISAba insertion elements results in high-level resistance to colistin in A. baumannii. In the present study, we quantify the rate of spontaneous insertional inactivation of LOS biosynthesis genes by ISAba elements in the ATCC 19606-type strain and two multidrug clinical isolates. Using insertional inactivation of lpxC by ISAba11 in the ATCC 19606 strain as a model, we determine the effect of several subinhibitory concentrations of the antibiotics, namely tetracycline, ciprofloxacin, meropenem, kanamycin and rifampicin, as well as the disinfectants ethanol and chlorhexidine on ISAba11 insertion frequencies. Notably, subinhibitory concentrations of tetracycline significantly increased ISAba11 insertion, and rifampicin completely inhibited the emergence of colistin resistance due to ISAba11 inactivation of lpxC. Sequencing of ISAba11 insertion sites within the lpxC gene demonstrated that insertions clustered between nucleotides 382 and 618 (58.3% of unique insertions detected), indicating that this may be a hotspot for ISAba11 insertion. The alignment of insertion sites revealed a semi-conserved AT-rich consensus sequence upstream of the ISAba11 insertion site, suggesting that ISAba11 insertion sites may be sequence-dependent. This study explores previously uncharacterized aspects regarding the acquisition of colistin resistance through insertional activation in LOS biosynthesis genes in A. baumannii.

Bacterial resistance to CRISPR-Cas antimicrobials

In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations. We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.

Analysis of blaCHDL Genes and Insertion Sequences Related to Carbapenem Resistance in Acinetobacter baumannii Clinical Strains Isolated in Warsaw, Poland

Acinetobacter baumannii is an important cause of nosocomial infections worldwide. The elucidation of the carbapenem resistance mechanisms of hospital strains is necessary for the effective treatment and prevention of resistance gene transmission. The main mechanism of carbapenem resistance in A. baumannii is carbapenemases, whose expressions are affected by the presence of insertion sequences (ISs) upstream of blaCHDL genes. In this study, 61 imipenem-nonsusceptible A. baumannii isolates were characterized using phenotypic (drug-susceptibility profile using CarbaAcineto NP) and molecular methods. Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were utilized for the genotyping. The majority of isolates (59/61) carried one of the following acquired blaCHDL genes: blaOXA-24-like (39/59), ISAba1-blaOXA-23-like (14/59) or ISAba3-blaOXA-58-like (6/59). Whole genome sequence analysis of 15 selected isolates identified the following intrinsic blaOXA-66 (OXA-51-like; n = 15) and acquired class D β-lactamases (CHDLs): ISAba1-blaOXA-23 (OXA-23-like; n = 7), ISAba3-blaOXA-58-ISAba3 (OXA-58-like; n = 2) and blaOXA-72 (OXA-24-like; n = 6). The isolates were classified into 21 pulsotypes using PFGE, and the representative 15 isolates were found to belong to sequence type ST2 of the Pasteur MLST scheme from the global IC2 clone. The Oxford MLST scheme revealed the diversity among these studied isolates, and identified five sequence types (ST195, ST208, ST208/ST1806, ST348 and ST425). CHDL-type carbapenemases and insertion elements upstream of the blaCHDL genes were found to be widespread among Polish A. baumannii clinical isolates, and this contributed to their carbapenem resistance.

Comparative genomic and phenotypic characterization of invasive non-typhoidal Salmonella isolates from Siaya, Kenya

Non-typhoidal Salmonella (NTS) is a major global health concern that often causes bloodstream infections in areas of the world affected by malnutrition and comorbidities such as HIV and malaria. Developing a strategy to control the emergence and spread of highly invasive and antimicrobial resistant NTS isolates requires a comprehensive analysis of epidemiological factors and molecular pathogenesis. Here, we characterize 11 NTS isolates that caused bloodstream infections in pediatric patients in Siaya, Kenya from 2003–2010. Nine isolates were identified as S. Typhimurium sequence type 313 while the other two were S. Enteritidis. Comprehensive genotypic and phenotypic analyses were performed to compare these isolates to those previously identified in sub-Saharan Africa. We identified a S. Typhimurium isolate referred to as UGA14 that displayed novel plasmid, pseudogene and resistance features as compared to other isolates reported from Africa. Notably, UGA14 is able to ferment both lactose and sucrose due to the acquisition of insertion elements on the pKST313 plasmid. These findings show for the first time the co-evolution of plasmid-mediated lactose and sucrose metabolism along with cephalosporin resistance in NTS further elucidating the evolutionary mechanisms of invasive NTS phenotypes. These results further support the use of combined genomic and phenotypic approaches to detect and characterize atypical NTS isolates in order to advance biosurveillance efforts that inform countermeasures aimed at controlling invasive and antimicrobial resistant NTS.

FENOMENA BILINGUALISME (CAMPUR-ALIH KODE) BAHASA DALAM PERCAKAPAN SANTRI DI PONDOK PESANTREN AL MANSHUR POPONGAN KLATEN

This study aims to discuss one of the phenomena of bilingualism (code switching) and interference as well as the factors causing the phenomenon of language bilingualism in santri conversations in Islamic boarding schools. The forms of bilingualism and interference include Javanese, Indonesian and Arabic. The approach in this research is descriptive qualitative which describes the state of the object of research based on the facts that appear as they are. The subjects of this study were the students of Al Manshur Popongan Klaten. Data collection techniques in the form of observation, interviews and documentation. The analysis technique during the field uses the model of Miles and Huberman, namely data collection, data reduction, data presentation and conclusion or verification. The results of the study note that there are 55 conversational data in the Al Manshur Popongan Islamic boarding school classified as follows; internal code 7 data transfer, external code 10 data transfer. Mix codes in linguistic elements that occur namely mixed code insertion of 12 data tangible elements, mixed code 5 data tangible codes, mixed code insertion elements in clause 7 data and mixed code insertion elements in the 5 word repetition element. Factors causing code switching and code mixing are the presence of speakers, interlocutors, prestige, changes in topic of conversation, identification of social roles, the desire to explain, and the habit of speakers.

Characterization of Three Porcine Acinetobacter towneri Strains Co-Harboring tet(X3) and blaOXA-58

Tigecycline is the antibiotic of last resort for the treatment of extensively drug-resistant bacterial infections, mainly those of multidrug-resistant Gram-negative bacteria. The plasmid-mediated tet(X3) gene has recently been described in various pathogens that are resistant to tigecycline. We report three tigecycline-resistant Acinetobacter towneri strains isolated from porcine faeces in China, which all contained the tet(X3)-harboring plasmids. A broth microdilution method was used to examine the antimicrobial susceptibility of the isolates, and S1-Nuclease digestion pulsed-field gel electrophoresis (S1-PFGE) was used to characterize their plasmid profiles. The whole-genome sequences of the isolates were determined with the Nanopore PromethION platform. The sequence analysis indicated that the strains were A. towneri. They showed resistance to multiple antibiotics, and all the resistance genes were located on plasmids. The three tet(X3)-harboring plasmids had a similar backbone structure, and all contained blaOXA-58 with various insertion elements (IS). ISCR2 is considered an important factor in tet(X3) mobilization. In addition to ISCR2, we demonstrate that IS26 generates a circular intermediate containing the tet(X3) gene, which could increase the dissemination risk. To our knowledge, this is the first report of tet(X3)- and blaOXA-58-harboring plasmids in A. towneri. Because the IS26 is frequently found in front of tet(X3), research should be directed toward the action of IS26 in the spread of tet(X3).

Snapshot Study of Whole Genome Sequences of Escherichia coli from Healthy Companion Animals, Livestock, Wildlife, Humans and Food in Italy

Animals, humans and food are all interconnected sources of antimicrobial resistance (AMR), allowing extensive and rapid exchange of AMR bacteria and genes. Whole genome sequencing (WGS) was used to characterize 279 Escherichia coli isolates obtained from animals (livestock, companion animals, wildlife), food and humans in Italy. E. coli predominantly belonged to commensal phylogroups B1 (46.6%) and A (29%) using the original Clermont criteria. One hundred and thirty-six sequence types (STs) were observed, including different pandemic (ST69, ST95, ST131) and emerging (ST10, ST23, ST58, ST117, ST405, ST648) extraintestinal pathogenic Escherichia coli (ExPEC) lineages. Eight antimicrobial resistance genes (ARGs) and five chromosomal mutations conferring resistance to highest priority critically important antimicrobials (HP-CIAs) were identified (qnrS1, qnrB19, mcr-1, blaCTX-M1,15,55, blaCMY-2, gyrA/parC/parE, ampC and pmrB). Twenty-two class 1 integron arrangements in 34 strains were characterized and 11 ARGs were designated as intI1 related gene cassettes (aadA1, aadA2, aadA5, aad23, ant2_Ia, dfrA1, dfrA7, dfrA14, dfrA12, dfrA17, cmlA1). Notably, most intI1 positive strains belonged to rabbit (38%) and poultry (24%) sources. Three rabbit samples carried the mcr-1 colistin resistance gene in association with IS6 family insertion elements. Poultry meat harbored some of the most prominent ExPEC STs, including ST131, ST69, ST10, ST23, and ST117. Wildlife showed a high average number of virulence-associated genes (VAGs) (mean = 10), mostly associated with an ExPEC pathotype and some predominant ExPEC lineages (ST23, ST117, ST648) were identified.

Prevalence and distribution of resistance and enterotoxins/enterotoxin-like genes in different clinical isolates of coagulase-negative Staphylococcus

Background Coagulase-negative staphylococcus (CoNS) is considered to be the major reservoirs for genes facilitating the evolution of S. aureus as a successful pathogen. The present study aimed to determine the occurrence of genes conferring resistance to fluoroquinolone, determining of the prevalence of insertion sequence elements IS256, IS257 and different superantigens (SAgs) among CoNS isolates obtained from various clinical sources. Materials and methods The current study conducted on a total of the 91 CoNS species recovered from clinical specimens in Hamadan hospitals in western Iran in 2017–2019. The antimicrobial susceptibility testing was performed using disk diffusion method and the presence of the IS256 and IS257, genes conferring resistance to fluoroquinolone and enterotoxins/enterotoxin-like encoding genes were investigated by polymerase chain reaction (PCR) method. Results Among genes encoding classic enterotoxins, sec was the most frequent which was carried by 48.4% of the 91 isolates, followed by seb in 27.5% of the isolates. None of the CoNS isolates was found to be positive to enterotoxin-like encoding genes. In 11(12%) of all isolates that were phenotypically resistant to levofloxacin, 9 isolates (81.8%) were positive for gyrB, 8 isolates (72.7%) were positive for gyrA, 8 isolates (72.7%) harbored grlB and 7 isolates (63.6%) were found to carry grlA. The IS256 and IS257 were identified in 31.8% and 74.7% of the isolates, respectively. The results of statistical analysis showed a significant association between the occurrence of staphylococcal enterotoxins (SEs) encoding genes and antimicrobial resistance. Conclusion Antimicrobial resistant determinants and SEs are co-present in clinical CoNS isolates that confer selective advantage for colonization and survival in hospital settings. The coexistence of insertion elements and antibiotic resistance indicate their role in pathogenesis and infectious diseases.