Autolysin as a fibronectin receptor on the cell surface of Clostridium perfringens.

OBJECTIVE: Clostridium perfringens causes food poisoning and gas gangrene, a serious wound-associated infection. C. perfringens cells adhere to collagen via fibronectin (Fn). We investigated whether the peptidoglycan hydrolase of C. perfringens, i.e., autolysin (Acp), is implicated in Fn binding to C. perfringens cells. METHODS: This study used recombinant Acp fragments, human Fn and knockout mutants (C. perfringens 13 acp::erm and HN13 ΔfbpC ΔfbpD). Ligand blotting, Western blotting analysis, and complementation tests were performed. The Fn-binding activity of each mutant was evaluated by ELISA. RESULTS: From an Fn-binding assay using recombinant Acp fragments, Fn was found to bind to the catalytic domain of Acp. In mutant cells lacking Acp, Fn binding was significantly decreased, but was restored by the complementation of the acp gene. There are three known kinds of Fn-binding proteins in C. perfringens: FbpC, FbpD, and glyceraldehyde-3-phosphate dehydrogenase. We found no difference in Fn-binding activity between the mutant cells lacking both FbpC and FbpD (SAK3 cells) and the wild-type cells, indicating that these Fn-binding proteins are not involved in Fn binding to C. perfringens cells. CONCLUSIONS: We found that the Acp is an Fn-binding protein that acts as an Fn receptor on the surface of C. perfringens cells.

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.

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.

Structural and biochemical characterization of the Clostridium perfringens-specific Zn2+-dependent amidase endolysin, Psa, catalytic domain.

Phage-derived endolysins, enzymes that degrade peptidoglycans, have the potential to serve as alternative antimicrobial agents. Psa, which was identified as an endolysin encoded in the genome of Clostridium perfringens st13, was shown to specifically lyse C. perfringens. Psa has an N-terminal catalytic domain that is homologous to the Amidase_2 domain (PF01510), and a novel C-terminal cell wall-binding domain. Here, we determined the X-ray structure of the Psa catalytic domain (Psa-CD) at 1.65 Å resolution. Psa-CD has a typical Amidase_2 domain structure, consisting of a spherical structure with a central ß-sheet surrounded by two α-helix groups. Furthermore, there is a Zn2+ at the center of Psa-CD catalytic reaction site, as well as a unique T-shaped substrate-binding groove consisting of two grooves on the molecule surface. We performed modeling study of the enzyme/substrate complex along with a mutational analysis, and demonstrated that the structure of the substrate-binding groove is closely related to the amidase activity. Furthermore, we proposed a Zn2+-mediated catalytic reaction mechanism for the Amidase_2 family, in which tyrosine constitutes part of the catalytic reaction site.

X-ray structures of Clostridium perfringens sortase C with C-terminal cell wall sorting motif of LPST demonstrate role of subsite for substrate-binding and structural variations of catalytic site.

Pili of Gram-positive bacteria are flexible rod proteins covalently attached to the bacterial cell wall, that play important roles in the initial adhesion of bacterial cells to host tissues and bacterial colonization. Pili are formed by the polymerization of major and minor pilins, catalyzed by class C sortase (SrtC), a family of cysteine transpeptidases. The Gram-positive bacterium Clostridium perfringens has a major pilin (CppA), a minor pilin (CppB), and SrtC (CpSrtC). CpSrtC recognizes the C-terminal cell wall sorting signal motifs with five amino acid residues, LPSTG of CppA and LPETG of CppB, for the polymerization of pili. Here, we report biochemical analysis to detect the formation of Clostridium perfringens pili in vivo, and the X-ray structure of a novel intermolecular substrate-enzyme complex of CpSrtC with a sequence of LPST at the C-terminal site. The results showed that CpSrtC has a subsite for substrate-binding to aid polymerization of pili, and that the catalytic site has structural variations, giving insights into the enzyme catalytic reaction mechanism and affinities for the C-terminal cell wall sorting signal motif sequences.

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.

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.

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.

First Genomic Characterization of blaVIM-1 and mcr-9-Coharbouring Enterobacter hormaechei Isolated from Food of Animal Origin.

We describe here the complete genome sequence of an Enterobacter hormaechei ST279 coharbouring blaVIM-1 and mcr-9 recovered from uncooked beef patty in June 2017, Egypt. The tested isolate was resistant to carbapenem but susceptible to colistin (minimum inhibitory concentration (MIC), 0.5 µg/mL). The antimicrobial susceptibility profile and conjugation experiments were performed. The entire genome was sequenced by the Illumina MiniSeq and Oxford Nanopore methods. The blaVIM-1 and mcr-9 genes are carried on the same IncHI2/pMLST1 plasmid, pMS37a (Size of 270.9 kb). The mcr-9 gene was located within the physical boundaries demarcated by two insertion elements IS903 (upstream) and IS1 (downstream) but did not possess the downstream regulatory genes (qseC/qseB) which regulate the expression of mcr-9. Therefore, the mcr-9 might be silently disseminated among carbapenem-resistant Enterobacterales. In addition to blaVIM-1 and mcr-9, plasmid pMS37a harbored various antibiotic resistance genes including aac(6')-Il, ΔaadA22, aac(6')-Ib-cr, sul1, dfrA1 and tetA. To the best of our knowledge, this is the first report of a blaVIM-1 and mcr-9-coharbouring E. hormaechei isolate of food origin worldwide. The identification of a multidrug-resistant VIM-1 and mcr-9 positive Enterobacter hormaechei isolate from food is worrisome as retail meat and meat products could serve as a vehicle for these MDR bacteria, which could be transferred between animals and humans through the food chain. It further highlights that Enterobacterales co-producing MCR and carbapenemases being found in the food chain indeed correspond to a One-Health issue, highlighting the need for serious steps to prevent their further dissemination.

Draft genome sequence of a blaNDM-1- and blaOXA-244-carrying multidrug-resistant Escherichia coli D-ST69 clinical isolate from Egypt.

OBJECTIVES: This study describes the first draft genome sequence of a multidrug-resistant (MDR) Escherichia coli D-ST69 clinical isolate from Egypt carrying blaNDM-1 and blaOXA-244. METHODS: The strain was isolated in December 2014 from a wound pus swab of a male patient in the city of Kafr El-Sheikh using MacConkey agar containing 2 µg/mL meropenem. The strain was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole-genome sequencing using an Illumina MiSeq platform. RESULTS: The draft genome of the strain (HR14_AS) was 5.08 Mbp in size containing a total of 90 contigs encoding 4677 predicted genes with an average G+C content of 50.7%. Strain HR14_AS belongs to sequence type 69 (ST69), phylogroup D and exhibits an MDR phenotype, with minimum inhibitory concentrations (MICs) of 64 µg/mL and 32 µg/mL for meropenem and doripenem, respectively. Multiple acquired antimicrobial resistance genes conferring resistance to macrolides [mdf(A)], fluoroquinolones [aac(6')-Ib-cr], quinolones (qnrS1), trimethoprim (dfrA14), ß-lactams (blaNDM-1, blaOXA-244, blaCTX-M-15, blaOXA-9 and blaTEM-1B) and aminoglycosides [aac(3)-IId, aac(6')-Ib, aadA1 and aph(3')-VI] were detected. The blaOXA-244 and blaNDM-1 genes were located on the chromosome (Tn6237) and on an IncI1-type self-conjugative plasmid of >93 kb in size, respectively. CONCLUSIONS: Here we report the first draft genome sequence of a MDR E. coli D-ST69 isolate carrying blaNDM-1 and blaOXA-244. Besides clonal expansion of the E. coli ST38 pandemic clone, this study further identified that the spread of OXA-244-producing E. coli could be related to mobilisation of the IS1R-made composite transposon (Tn6237) carrying blaOXA-244.

Emergence of a Multidrug-Resistant Enterobacter hormaechei Clinical Isolate from Egypt Co-Harboring mcr-9 and blaVIM-4.

Abstract: This study describes the first full genomic sequence of an mcr-9 and blaVIM-4-carrying multidrug-resistant Enterobacter hormaechei clinical isolate from Egypt. The strain was isolated in April 2015 from the sputum of a patient in Cairo, Egypt. The mcr-9 and blaVIM-4 genes were identified by PCR screening and DNA sequencing; the isolate was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole genomic sequencing. mcr-9 and blaVIM-4 were carried by an IncHI2 plasmid, pAMS-38a (281,121 bp in size); the plasmid also carried genes conferring resistance against sulfonamides (sul1), quinolones (qnrA1), trimethoprim (dfrA1), ß-lactams (blaTEM-1B), aminoglycosides (aac (6')-II, aadA23, aadA2b, and ant(2'')-Ia). The strain was susceptible to colistin (MIC, <0.25 µg/mL); this could be due to the absence of the qseC/qseB regulatory system located downstream of mcr-9 in Enterobacterales, which is involved in the induction of colistin-resistance. The genetic context of mcr-9 and blaVIM-4 was identified as IS1-mcr-9-IS903-pcoS-∆pcoE-rcnA and intI1-blaVIM-4-aac (6')-II-dfrA1-∆aadA23-smr-ISPa21-qacE∆1, respectively. This is the first report of an mcr-9 and blaVIM-4 /IncHI2-carrying multidrug-resistant E. hormaechei clinical isolate from Africa and the Middle East. Plasmids of the IncHI2 group and the two insertion sequences (IS1, and IS903) might be the main vehicles for dissemination of mcr-9. Further screening for mcr-9 is essential for identifying its incidence and to prevent its dissemination.

Draft genome sequence of an mcr-1/IncI2-carrying multidrug-resistant Escherichia coli B1:ST101 isolated from meat and meat products in Egypt.

OBJECTIVES: The aim of this study was to investigate the occurrence of plasmid-encoded colistin resistance among Gram-negative bacteria isolated from meat and meat products in Egypt and to report the draft genome sequence of anmcr-1/IncI2-carrying multidrug-resistant (MDR) Escherichia coli B1:ST101 isolate. METHODS: A total of 128 colistin-resistant strains were isolated from various meat and meat product samples in different cities in Egypt. Multiplex PCR screening for plasmid-mediated colistin resistance genes was performed. Whole-genome sequencing was performed using an Illumina NextSeq platform and the genome was assembled using CLC Genomics Workbench 7.5.1. RESULTS: A singlemcr-1-positive MDR E. coli strain was isolated from beef sausages. The genome size of the E. coli strain was calculated at 5 044 715bp, with a total of 226 contigs and a G+C content of 50.5%. The strain belonged to ST101 (phylogroup B1). The mcr-1 gene was located on an IncI2-type self-conjugative plasmid of 64.6kb in size. The strain showed a MDR phenotype, with a colistin MIC of 4mg/L. A large number of acquired antimicrobial resistance genes was identified, including genes encoding resistance to colistin (mcr-1), ß-lactams (blaTEM-1), phenicols (floR), trimethoprim (dfrA12), aminoglycosides [aac(3)-IIa, aph(3")-Ib and aadA2], macrolides (mphA and mdfA), tetracyclines (tetA), sulfonamides (sul1 and sul2) and quinolones (qnrS1). CONCLUSION: Here we report the first draft genome sequence of anmcr-1/IncI2-carrying MDR E. coli B1:ST101 isolated from beef sausage in Egypt. This study highlights the potential role played by food products in the spread of colistin resistance to humans.

Genetic analysis of carbapenemase-producing Gram-negative bacteria isolated from a university teaching hospital in Egypt.

A total of 65 non-replicate Gram-negative bacterial strains were recovered from clinical specimens between April and September 2014 at a University Hospital in Egypt. This collection was screened by PCR for carbapenemase-encoding genes, 16S rRNA methylases, and colistin resistance genes (mcr-1-mcr-8). Twenty-two strains (22/65, 33.8%) were positive for carbapenemase-encoding genes [13 NDM-1-producers (four Escherichia coli, two Klebsiella pneumoniae, and seven Providencia stuartii), two E. coli co-carrying NDM-5 and OXA-181, and seven Pseudomonas aeruginosa (three VIM-2, four VIM-24) strains]. The 16S rRNA methylase RmtC was detected in 12 NDM-1-producers for the first time in Egypt; no mcr genes were detected. A self-transmissible A/C plasmid was found to carry blaNDM-1 in all NDM-1-producing strains. NDM-5 and OXA-181 were located on an untypeable and IncX3 plasmid, respectively. Additionally, Enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed five clonally related P. stuartii isolates collected over a 1.5-month period. Thirteen carbapenemase-producing strains were isolated from burn patients who are at a high risk of developing infections and require special medical care. To our knowledge, this is the first report of NDM-1-producing-P. stuartii strains in an African burn unit, NDM-1- and RmtC-positive non-lactose fermenting E. coli globally, VIM-24-producing P. aeruginosa in Africa, and 16S RMTase rmtC-NDM-1-producers in Egypt. This work highlights the detection of different carbapenemase-producing bacterial strains within an Egyptian teaching hospital compromising the effectiveness of carbapenems and urgently asking the Egyptian medical authorities for implementation of antimicrobial surveillance plans and infection control policies to early detect and to effectively halt the rapid spread of these superbugs.

Structures of major pilins in Clostridium perfringens demonstrate dynamic conformational change.

Pili in Gram-positive bacteria are flexible rod proteins associated with the bacterial cell surface, and they play important roles in the initial adhesion to host tissues and colonization. The pilus shaft is formed by the covalent polymerization of major pilins, catalyzed by sortases, a family of cysteine transpeptidases. Here, X-ray structures of the major pilins from Clostridium perfringens strains 13 and SM101 and of sortase from strain SM101 are presented with biochemical analysis to detect the formation of pili in vivo. The major pilin from strain 13 adopts an elongated structure to form noncovalently linked polymeric chains in the crystal, yielding a practical model of the pilus fiber structure. The major pilin from strain SM101 adopts a novel bent structure and associates to form a left-handed twist like an antiparallel double helix in the crystal, which is likely to promote bacterial cell-cell interactions. A modeling study showed that pilin with a bent structure interacts favorably with sortase. The major pilin from strain SM101 was considered to be in an equilibrium state between an elongated and a bent structure through dynamic conformational change, which may be involved in pili-mediated colonization and sortase-mediated polymerization of pili.

High Prevalence of Antimicrobial Resistance in Gram-Negative Bacteria Isolated from Clinical Settings in Egypt: Recalling for Judicious Use of Conventional Antimicrobials in Developing Nations.

This study was designed to investigate, at the molecular level, the antimicrobial resistance mechanisms of different antimicrobial resistance genes, including, extended-spectrum ß-lactamases, AmpC ß-lactamases, class 1 and 2 integrons, and plasmid-mediated quinolone resistance genes of Gram-negative bacteria isolated from clinical settings in Egypt. A total of 126 nonduplicate Gram-negative isolates were recovered from different clinical samples taken from hospitalized patients in Egypt in 2014. Antimicrobial susceptibility testing showed that, 93.6% (118/126) of the isolates had a multidrug-resistant phenotype. Interestingly, we reported a high level of antimicrobial resistance nearly for all tested antibiotics; to our knowledge, this is the first report from Egypt indicating very high level of antibiotic resistance in Egypt. Polymerase chain reaction screening and DNA sequencing revealed that, 75.4% (95/126) of the isolates harbored at least one extended-spectrum ß-lactamase-encoding gene, with blaCTX-M being the most prevalent (65.9%), followed by blaSHV (46.8%). The AmpC ß-lactamase, blaCMY, was detected in 7.1% (9/126) of bacterial isolates, with blaCMY-42 being the most prevalent. Class 1 integrons were detected in 50.8% (64/126) of the isolates, and class 2 integrons were detected in 2.4% (3/126) of the isolates. The plasmid-mediated quinolone resistance gene, qnr, was detected in 58.7% (74/126) of the tested isolates, with qnrS being the most prevalent. Several antimicrobial resistance determinants were identified in Egypt for the first time, such as SHV-27, SHV-28, SHV-33, SHV-63, SHV-71, SHV-82, SHV-142, CMY-42, CMY-6, and the new CMY-72 like. This study highlights the importance of the conscious use of conventional antimicrobials to overcome the multidrug resistance problem.

Systemic method to isolate large bacteriophages for use in biocontrol of a wide-range of pathogenic bacteria.

Large phages are characterized by genomes around 200 kbp or more. They can infect wide host ranges of bacteria and maintain long-lasting infection. There is no standard method for selective isolation of large phages. In this study, we developed a systemic method to isolate large phages and succeeded in isolating 11 large phages, named Escherichia phage E1∼E11. Electron microscopy observations revealed typical Myoviridae phages with big capsids and long contractile tails. Genome sizes of the isolated phages were determined by pulsed-field gel electrophoresis and found to be in two groups, those around 200 kbp for E1, E2, E5, E6, E7, E9 and E10 phages, and others of approximately 450 kbp for E3, E4, E8 and E11 phages. The isolated large phages had wide host ranges: for example, E9 was effective against Shigella sonnei SH05001, Shigella bydii SH00007, Shigella flexneri SH00006, Salmonella enterica serovar Enteritidis SAL01078 and Escherichia coli C3000 (K-12 derivative), as well as its original host E. coli BL21. Screening of these jumbo phages was performed with non-pathogenic E. coli strains as hosts. Therefore, this method opens a way to isolate jumbo phages infecting wide ranges of pathogenic bacteria in a typical laboratory with standard laboratory strains as the hosts. The isolated large phages will be good candidates for biocontrol of various pathogens.

An enhanced green fluorescence protein (EGFP)-based reporter assay for quantitative detection of sporulation in Clostridium perfringens SM101.

Clostridium perfringens type F is a spore-forming anaerobe that causes bacterial food-borne illness in humans. The disease develops when ingested vegetative cells reach the intestinal tract and begin to form spores that produce the diarrheagenic C. perfringens enterotoxin (CPE). Given that CPE production is regulated by the master regulator of sporulation (transcription factor Spo0A), the identification of sporulation-inducing factors in the intestine is relevant to better understanding of the disease. To examine these factors, we established assays to quantify C. perfringens sporulation stage under microscopy by using two fluorescent reporters, namely, Evoglow-Bs2 and CpEGFP. When the reporter genes were placed under control of the cpe promoter, both protein products were expressed specifically during sporulation. However, the intensity of the anaerobic reporter Evoglow-Bs2 was weak and rapidly photobleached during microscopic observation. Alternatively, CpEGFP, a canonical green fluorescence protein with optimized codon usage for Clostridium species, was readily detectable in the mother-cell compartment of most bacteria at early stages of sporulation. Additionally, CpEGFP expression predicted final spore yield and was quantifiable in 96-well plates using fluorescence plate reader. These results indicate that CpEGFP can be used to analyze the sporulation of C. perfringens and has a potential application in the large-scale screening of sporulation-regulating biomolecules.