Sequence Polymorphisms in Vibrio cholerae HapR Affect Biofilm Formation under Aerobic and Anaerobic Conditions.

We investigated the influence of hapR sequence mutations on the biofilm formation of Vibrio cholerae. In this study, hapR sequences from 85 V. cholerae strains belonging to both pandemic and nonpandemic serogroup were investigated through phylogenetic and sequence analyses. Biofilm formation assays under aerobic and anaerobic conditions were also performed. Sequence variations include single point mutations and insertions/deletions (indels) leading to either truncated or frameshifted HapR. Population structure analysis revealed two major hapR haplogroups, hapR1 and hapR2. Phylogenetic reconstruction displayed a hypothetical ancestral hapR sequence located within the hapR1 haplogroup. Higher numbers of single nucleotide polymorphisms and genetic diversity indices were observed in hapR1, while indels occurred dominantly in hapR2. Aerobic conditions supported more robust biofilms compared to anaerobic conditions. Strains with frameshifted HapR produced the largest amount of biofilm under both oxygen conditions. Quantitative real-time PCR assay confirmed that strains with truncated and frameshifted HapR resulted in a nonfunctional regulator as exhibited by the significantly low hapA gene expression. The present study shows that HapR mutations had a strong influence on biofilm formation and that sequence polymorphisms leading to the disruption of DNA-binding sites or dimerization of the HapR will result in more-robust V. cholerae biofilms. IMPORTANCE Our study revealed an ancestral hapR sequence from a phylogenetic reconstruction that displayed the evolutionary lineage of the nonpandemic to the pandemic strains. Here, we established hapR1 and hapR2 as major hapR haplogroups. The association of the O1 and O139 serogroups with the hapR2 haplogroup demonstrated the distinction of hapR2 in causing cholera infection. Moreover, mutations in this regulator that could lead to the disruption of transcription factor-binding sites or dimerization of the HapR can significantly affect the biofilm formation of V. cholerae. These observations on the relationship of the hapR polymorphism and V. cholerae biofilm formation will provide additional considerations for future biofilm studies and insights into the epidemiology of the pathogen that could ultimately help in the surveillance and mitigation of future cholera disease outbreaks.

Coproduction of Tet(X7) Conferring High-Level Tigecycline Resistance, Fosfomycin FosA4, and Colistin Mcr-1.1 in Escherichia coli Strains from Chickens in Egypt.

The plasmid-mediated tet(X7) conferring high-level tigecycline resistance was identified in five mcr-1.1-positive Escherichia coli strains (ST10 [n = 3] and ST155 [n = 2]) isolated from chickens in Egypt. Two fosfomycin-resistant fosA4-carrying IncFII plasmids (∼79 kb in size) were detected. Transposase ISCR3 (IS91 family) is syntenic with tet(X7) in all isolates, suggesting its role in the mobilization of tet(X7). To our knowledge, this is the first global report of ST4-IncHI2 plasmids cocarrying tet(X7) and mcr-1.1 from chickens.

Vegetable-Derived Carbapenemase-Producing High-Risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 Clones in Japan: Coexistence of blaNDM-1, blaOXA-66, blaOXA-72, and an AbaR4-Like Resistance Island in the Same Sample.

This study was conducted to characterize carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumannii isolated from fresh vegetables in Japan. Two K. pneumoniae isolates (AO15 and AO22) and one A. baumannii isolate (AO22) were collected from vegetables in the city of Higashihiroshima, Japan, and subjected to antimicrobial susceptibility testing, conjugation experiments, and complete genome sequencing using Illumina MiniSeq and Oxford Nanopore MinION sequencing platforms. The two K. pneumoniae isolates were clonal, belonging to sequence type 15 (ST15), and were determined to carry 19 different antimicrobial resistance genes, including blaNDM-1 Both the isolates carried blaNDM-1 on a self-transmissible IncFII(K):IncR plasmid of 122,804 bp with other genes conferring resistance to aminoglycosides [aac(6')-Ib, aadA1, and aph(3')-VI], ß-lactams (blaCTX-M-15, blaOXA-9, and blaTEM-1A), fluoroquinolones [aac(6')-Ib-cr], and quinolones (qnrS1). A. baumannii AO22 carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid of 10,880 bp. Interestingly, A. baumannii AO22 harbored an AbaR4-like genomic resistance island (GI) of 41,665 bp carrying genes conferring resistance to tetracycline [tet(B)], sulfonamides (sul2), and streptomycin (strAB). Here, we identified Japanese carbapenemase-producing Gram-negative bacteria isolated from vegetables, posing a food safety issue and a public health concern. Additionally, we reported a GR2-type plasmid carrying two copies of blaOXA-72 and an AbaR4-like resistance island from a foodborne A. baumannii isolate.IMPORTANCE Carbapenemase-producing Gram-negative bacteria (CPGNB) cause severe health care-associated infections and constitute a major public health threat. Here, we investigated the genetic features of CPGNB isolated from fresh vegetable samples in Japan and found CPGNB, including Klebsiella pneumoniae and Acinetobacter baumannii, with dissimilar carbapenemases. The NDM carbapenemase, rarely described in Japan, was detected in two K. pneumoniae isolates. The A. baumannii isolate identified in this study carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid. This study indicates that even one fresh ready-to-eat vegetable sample might serve as a significant source of genes (blaNDM-1, blaOXA-72, blaCTX-M-14b, and blaCTX-M-15) encoding resistance to frontline and clinically important antibiotics (carbapenems and cephalosporins). Furthermore, the detection of these organisms in fresh vegetables in Japan is alarming and poses a food safety issue and a public health concern.

Sphingolipids regulate telomere clustering by affecting the transcription of genes involved in telomere homeostasis.

In eukaryotic organisms, including mammals, nematodes and yeasts, the ends of chromosomes, telomeres are clustered at the nuclear periphery. Telomere clustering is assumed to be functionally important because proper organization of chromosomes is necessary for proper genome function and stability. However, the mechanisms and physiological roles of telomere clustering remain poorly understood. In this study, we demonstrate a role for sphingolipids in telomere clustering in the budding yeast Saccharomyces cerevisiae. Because abnormal sphingolipid metabolism causes downregulation of expression levels of genes involved in telomere organization, sphingolipids appear to control telomere clustering at the transcriptional level. In addition, the data presented here provide evidence that telomere clustering is required to protect chromosome ends from DNA-damage checkpoint signaling. As sphingolipids are found in all eukaryotes, we speculate that sphingolipid-based regulation of telomere clustering and the protective role of telomere clusters in maintaining genome stability might be conserved in eukaryotes.

Antimicrobial Effects of Blueberry, Raspberry, and Strawberry Aqueous Extracts and their Effects on Virulence Gene Expression in Vibrio cholerae.

The antimicrobial effects of aqueous extracts of blueberry, raspberry, and strawberry on 13 pathogenic bacteria were evaluated. The minimum inhibitory concentrations and minimum bactericidal concentrations of the extracts were determined before and after neutralization to pH 7.03 ± 0.15. Both Gram-positive and Gram-negative pathogenic bacteria were selectively inhibited by the non-neutralized berries. Blueberry was the best inhibitor, and Vibrio and Listeria were the most sensitive bacteria. After neutralization, blueberry affected only Vibrio and Listeria, whereas the antimicrobial activities of raspberry and strawberry were abolished. The total contents of phenolics, flavonoids, and proanthocyanidins in the extracts were measured with colorimetric methods and were highest in strawberry, followed by raspberry, and then blueberry. We also studied the effects of sub-bactericidal concentrations of the three berry extracts on virulence gene expression in Vibrio cholerae. Real-time quantitative reverse transcription-polymerase chain reaction revealed that the three berry extracts effectively repressed the transcription of the tcpA gene. Raspberry also repressed the transcription of the ctxA gene, whereas blueberry and strawberry did not. However, the three berry extracts did not affect the transcription of toxT. These results suggest that the three berry extracts exert potent antimicrobial effects and inhibit the expression of the virulence factors of V. cholerae.

Genetic characterization of antibiotic resistance and virulence factors in Enterococcus spp. from Japanese retail ready-to-eat raw fish.

Little information is available on the diversity and distribution of resistance and virulence factors in enterococci isolated from retail fish. In this study, 200 samples of retail ready-to-eat raw fish (sashimi) collected from the Japanese prefecture of Hiroshima were analyzed for incidence of Enterococcus spp. We recovered 96 enterococcal isolates from 90 (45%, 90/200) samples. Fifty-six strains were identified at the species level: E. faecalis (n = 31), E. faecium (n = 7), E. casseliflavus (n = 7), E. gallinarum (n = 3), E. phoeniculicola (n = 4), E. raffinosus (n = 2), E. saccharolyticus (n = 1), and E. gilvus (n = 1). Twenty-five (26%, 25/96) strains carried antibiotic resistance genes. These included the tet(M), tet(L), tet(K), erm(B), msr(A/B), aph(3'), and blaZ genes, which were detected in 12.5%, 9.3%, 2%, 14.5%, 1%, 1%, and 2% of isolates, respectively. The virulence genes gelE and asa1 were detected in 31 and 24 E. faecalis strains, respectively. Both genes were detected in one E. faecium strain. In conclusion, this is the first study to underscore the importance of sashimi as not only a reservoir of Enterococcus spp. carrying resistance and virulence genes, but also a reservoir for unusual Enterococcus spp.

Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from septicemic broilers.

Avian pathogenic Escherichia coli (APEC) causes extensive mortality in poultry flocks, leading to extensive economic losses. To date, little information is available on the molecular basis of antimicrobial resistance in APEC in Africa. Therefore, the objective of this study was to characterize the virulence and antimicrobial resistance of multidrug-resistant APEC isolated from septicemic broilers in Egypt at the molecular level. Among 91 non-repetitive E. coli isolates, 73 (80.2%) carried three or more of the APEC virulence genes iroN, ompT, iss, iutA, and hlyF. All 73 APEC isolates showed multidrug resistance phenotypes, particularly against ampicillin, tetracycline, spectinomycin, streptomycin, kanamycin, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 and class 2 integrons in 34 (46.6%) and seven (9.6%) isolates, respectively. The ß-lactamase-encoding genes, bla(TEM-1), bla(TEM-104), bla(CMY-2), bla(OXA-30), bla(CTX-M-15), and bla(SHV-2); tetracycline resistance genes, tet(A), tet(B), tet(C), tet(D), and tet(E); the plasmid-mediated quinolone resistance genes, qnrA1, qnrB2, qnrS1, and aac(6')-Ib-cr, and florfenicol resistance gene, floR, were also identified in 69 (94.5%), 67 (91.8%), 47 (64.4%), and 13 (17.8%) isolates, respectively. To the best of our knowledge, this is the first report of molecular characterization of antimicrobial resistance in APEC strains from Africa.

Predominance of Multidrug-Resistant Gram-Negative Bacteria Isolated from Supermarket Retail Seafood in Japan.

Reports have documented antimicrobial usage in aquaculture, and the aquatic ecosystem can be considered a genetic storage site for antibiotic-resistant bacteria. This study assessed the prevalence of antimicrobial resistance (AMR) among Gram-negative bacteria recovered from retail seafood in Hiroshima, Japan. A total of 412 bacteria were isolated and screened for the presence of ß-lactamases, acquired carbapenemases, and mobile colistin-resistance (mcr) genes. Forty-five (10.9%) isolates were dominated by Morganella (28%), Proteus (22%), Aeromonas (14%), Citrobacter (8%), and Escherichia (8%) and carried AMR genes. The identified AMR genes included those encoded in integrons (19), aac(6՛)-Ib (11), blaTEM-1 (7), blaCTX-M-like (12), blaCTX-M-65 (2), blaSHV-12 (1), blaSHV-27 (1), blaOXA-10 (1), blaOXA-2 (1), and mcr (2). The most common clinical resistances were against ampicillin, colistin, sulfamethoxazole/trimethoprim, tetracycline, and ciprofloxacin. Multidrug resistance (MDR) occurred in 27 (60%) AMR isolates, and multiple antibiotic resistance indices ranged from 0.2 to 0.8. A conjugation experiment showed that 10 of the 11 selected MDR strains harbored conjugable plasmids, although PCR-based replicon typing described seven strains as untypable. IncF replicon was identified in MDR extended-spectrum ß-lactamase-producing Escherichia coli of the pathogenic B2 phylogroup. Our findings suggest that retail seafood harbors MDR bacteria of human interest that require strict resistance surveillance in the seafood production continuum.

Comparative molecular profiling of antimicrobial resistance and phylogenetic characterization of multidrug-resistant Escherichia coli isolated from meat sources in 2009 and 2021 in Japan.

The global spread of antimicrobial resistance (AMR) is alarming. Escherichia coli is a Gram-negative bacterium that causes healthcare-associated infections and is a major threat to public health. Currently, no comprehensive antimicrobial surveillance of multidrug-resistant E. coli of diverse phylogroups along the meat value chain has been implemented in Higashihiroshima, Japan. Therefore, by employing the One Health approach, 1183 bacterial isolates, including 303 recovered from meat samples in 2009, were screened for the presence of antimicrobial resistance determinants using multiplex PCR and DNA sequencing techniques. Seventy-seven non-duplicate E. coli isolates that harbored AMR genes were subjected to antimicrobial susceptibility testing and the detection of integrons. Phylogenetic characterization, which has not been previously investigated, was used to assign E. coli to one of the eight phylogroups. Twenty-six out of 33 (78.8%) and 34 out of 44 (77.3%) E. coli isolates from 2009 and 2021 exhibited multidrug resistance (MDR) phenotypes, respectively. The most common clinical resistance was observed against ampicillin, tetracycline, kanamycin, sulfamethoxazole/trimethoprim, cefotaxime, and chloramphenicol. Overall, 22.1% (17/77) of the E. coli isolates carried extended-spectrum ß-lactamase (ESBL)-encoding genes and showed the ESBL-resistant phenotypes. For the two isolation years, AmpC/ESBL prevalence decreased from 42.4% in 2009 to 20.5% in 2021. The identified AMR genes included blaCTX-M-1, blaCTX-M-2, blaCTX-M-14, blaCTX-M-15, and blaSHV-12 (ESBL-types); blaSHV-1, blaTEM-1, blaTEM-135, and blaTEM-176 (narrow-spectrum types); blaCMY-4, blaADC-32, blaADC-216, blaACT-48, and blaACT-51 (AmpC types); and integrons. All E. coli isolates were negative for carbapenemase-encoding genes, whereas one isolate from 2009 carried mcr-5.1 allele. Approximately 52% of E. coli isolates identified in 2009 were assigned to phylogroup A compared to the 20.5% in 2021. Notably, the highest proportions of E. coli phylogroups exhibiting MDR were groups A, B1, and F, suggesting that members of these groups are mostly associated with drug resistance. This study highlights the role of meat as a significant reservoir of MDR E. coli and potential source for transmission of AMR genes. Our findings emphasize the importance of continuous monitoring to track the changes in the spread of antimicrobial resistance in the food chain.

A Novel Single-Tube Eicosaplex/Octaplex PCR System for the Detection of Extended-Spectrum ß-Lactamases, Plasmid-Mediated AmpC ß-Lactamases, and Integrons in Gram-Negative Bacteria.

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum ß-lactamases (ESBLs), plasmid-mediated AmpC ß-lactamases, aac(6')-Ib gene, and integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was blaRANH-2, a plasmid-mediated CTX-M related ESBL gene only found in Rahnella spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Pseudomonas-related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant ß-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC ß-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.

Emergence of colistin-resistant Enterobacter cloacae and Raoultella ornithinolytica carrying the phosphoethanolamine transferase gene, mcr-9, derived from vegetables in Japan.

IMPORTANCE: Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two mcr-9.1-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica. This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of mcr alleles.

Complete genome sequences of two Escherichia coli clinical isolates from Egypt carrying mcr-1 on IncP and IncX4 plasmids.

Colistin is a last-resort antibiotic used in the treatment of multidrug resistant Gram-negative bacteria. However, the activity and efficacy of colistin has been compromised by the worldwide spread of the mobile colistin resistance genes (mcr-1 to mcr-10). In this study, two clinical Escherichia coli strains, named EcCAI51, and EcCAI73, harbored mcr-1, showed multidrug-resistant phenotypes (with colistin MIC = 4 µg/ml), and belonged to phylogroup D: multilocus sequence type 1011 (ST1011) and phylogroup A: ST744, respectively. Findings revealed the existence of mcr-1 gene on two conjugable plasmids, pAMS-51-MCR1 (∼122 kb IncP) and pAMS-73-MCR1 (∼33 kb IncX4), in EcCAI51, and EcCAI73, respectively. The mcr-1-pap2 element was detected in the two plasmids. Additionally, the composite transposon (ISApl1-IS5D-pap2-mcr-1-ISApl1) was identified only in pAMS-51-MCR1 suggesting the potential for horizontal gene transfer. The two strains carried from 16 to 18 different multiple acquired antimicrobial resistance genes (ARGs). Additionally, two different multireplicon virulence plasmids (∼117 kb pAMS-51-Vr and ∼226 kb pAMS-73-Vr) carrying the sit operon, the Salmochelin siderophore iroBCDE operon and other several virulence genes were identified from the two strains. Hierarchical clustering of core genome MLST (HierCC) revealed clustering of EcCAI73, and EcCAI51 with global E. coli lineages at HC levels of 50 (HC50) to 100 (HC100) core genome allelic differences. To the best of our knowledge, this study presented the first complete genomic sequences of mcr-1-carrying IncP and IncX4 plasmids from human clinical E. coli isolates in Egypt. In addition, the study illustrated the mcr-1 broad dissemination in diverse plasmids and dissimilar E. coli clones.

Two novel retron elements are replaced with retron-Vc95 in Vibrio cholerae.

Bacterial reverse transcriptase (RT) is responsible for the production of an RNA-DNA complex called multicopy single-stranded DNA (msDNA). The genetic element required for the sysnthesis of msDNA is named a retron. Here, we characterize two novel retrons named retron-Vc81 and retron-Vc137 in Vibrio cholerae. Interestingly, retron-Vc81 and retron-Vc137 are replaced by retron-Vc95 at the same location on the chromosome.

Genetic Characterization of Carbapenemase-Producing Enterobacter cloacae Complex and Pseudomonas aeruginosa of Food of Animal Origin from Egypt.

The increasing spread of carbapenem resistance is a serious global public health concern that negatively affects human and animal health. In this study we characterized the carbapenemase production in gram-negative bacteria isolated from different meat and meat products in Egypt. Phenotypic and genotypic susceptibility testing were investigated. Two Enterobacter cloacae complex strains, isolated from kofta and beef burger, and one Pseudomonas aeruginosa isolated from minced meat, were found to harbor VIM-1 and VIM-2, respectively. These isolates showed multidrug resistance phenotype. The phenotypic carbapenemase production was confirmed with Carba NP test in addition to modified Hodge test, modified carbapenem inactivation method, and ethylenediaminetetraacetic acid inhibition test. The blaVIM-1 gene in both non-clonally related E. cloacae complex strains was part of a class 1 integron that also carried other resistance gene cassettes such as aacA7, dfrA1, ΔaadA, and smr. This integron was uncommonly disrupted by the insertion sequence ISPa21, located on a self-conjugative plasmid of either the A/C or HI2 incompatibility group with a size of >93 kb. The blaVIM-2 gene was identified within a class 1 integron, followed downstream by resistance genes aadB and blaOXA-10. The transfer of blaVIM-2 gene from P. aeruginosa failed, suggesting that this gene was located on the chromosome. Further studies are needed to screen the dissemination of carbapenemase-producing bacteria in both the environment and food chain.

A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm.

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme's specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.