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Background: Hypermucoviscous carbapenem-resistant Klebsiella pneumoniae (hmCRKp) is emerging globally and approaching the worst-case scenario in health care system. Aims: The main objective in this study was to determine the hypermucoviscous characteristics among the carbapenem-resistant K. pneumoniae (CRKp) isolated from a teaching hospital in Malaysia. The association of hypermucoviscous phenotype with the virulence traits and clinical presentations were also investigated. Methods: A retrospective study was conducted in University Malaya Medical Centre (UMMC). The presence of hypermucoviscous K. pneumoniae was identified among a collection of CRKp clinical isolates (first isolate per patient) from 2014 to 2015 using string test. Correlation between clinical and microbial characteristics of the hmCRKp was investigated. Results: A total of nine (7.5%) hmCRKp were detected among 120 CRKp isolates. Majority of the isolates were hospital acquired or health care-associated infections. None of the patients had typical pyogenic liver abscess. All of the hmCRKp isolates harbored carbapenemase genes and were multidrug resistant. K1/K serotype, peg-344, allS, and magA were not identified among hmCRKp isolates, whereas aerobactin siderophore receptor gene (iutA), iroB, rmpA, and rmpA2 were detected. Only three hmCRKp isolates were resistant to serum bactericidal. Conclusions: All the isolates presented inconclusive evidence for the interpretation of hypervirulence. Therefore, more study should be performed in the future to have a better understanding of the virulence mechanisms in correlation with the clinical and microbial determinants.

Objective Comparative phenotypic and genetic assessment of the pathogenic potential of E. coli strains isolated from patients with calculous pyelonephritis. Materials and methods 78 strains of E. coli isolated from urine of patients with calculous pyelonephritis in the acute phase (n=58) and in the remission phase (n=20). Escherichia were investigated for the presence of virulence genes papA, pap EF, papGII; afa, bma E, iutA, fyuA, feoB, kspMTII, usp multiplex PCR using selected primers. Phenotypically determined the ability to biofilm formation, antilysozyme, antihemoglobin, anticytokine, adhesive and sIgA-protease activity E. coli. Results The virulent potential of Escherichia coli at the pheno- and genotype levels was characterized. In strains of E. coli isolated from the urine of patients in the remission phase, the ability to form biofilms was more often and with high values of the trait; and in strains isolated in relapse - adhesive activity, the ability to inactivate pro- and anti-inflammatory cytokines, antihemoglobin activity, and genes encoding aphimbrial adhesin (afa), responsible for the synthesis of siderophore aerobactin (iutA), transporting bivalent iron (feoB). Conclusion The revealed differences in the pheno- and genotypic profiles between the cultures of Escherichia coli isolated from patients with calculous pyelonephritis in the phases of exacerbation and remission make it possible to differentiate the isolated strain and predict the course of the infectious-inflammatory process.

Background: K57 Klebsiella pneumoniae (K57-KP) is associated with hypervirulence, but the basis and systematic data of K57-KP are limited. Materials and Methods: A retrospective study was conducted in 156 patients between January 2013 and January 2016. The clinical and molecular data, including antimicrobial susceptibility testing, multilocus sequence typing, antimicrobial resistance genes, and virulence determinants were assessed. Results: Among the 39 K57-KP isolates, 14 isolates (35.9%) were associated with various types of invasive infections. Diabetes, drainage, use of carbapenems and quinolone antibiotics were dependent risk factors for K57-KP infections. Sequence type (ST)412 was the most prevalent among K57-KP isolates. K57-KP isolates were more resistant to clinically often used antimicrobial agents than hvKP (K1/K2) strains, and 12.8% (5/39) of the strains were resistant to carbapenems, which all harbored blaKPC-2. The prevalence of hypermucoviscosity phenotype, aerobactin, rmpA, rmpA2, and ybts revealed 66.7%, 100%, 89.7%, 89.7%, and 30.8%, whereas wcaG, allS, magA and kfu revealed 0%, 0%, 0%, and 5.1%, which were significantly lower than that of hvKP (K1/K2). The serum sensitivity, neutrophil phagocytic rate, and biofilm formation capacity of K57-KP strains were higher than that of K1/K2. Conclusion: There were no significant differences in the prevalence of hypermucoviscosity phenotype, carriage of rmpA and aerobactin genes between K57 and K1/K2 isolates, but the composition and production of capsule polysaccharide of K57-KP may be different from that of K1/K2 strains. K57-KP isolates exhibited distinctive virulence-associated traits, most of which belonged to ST412. Physicians should enhance the management of K57-KP infections because of the emergence of more and more carbapenem-resistant K57-KP isolates.

Purpose To characterize the clinical, resistance, and virulence features of carbapenem-resistant Klebsiella pneumonaie (CRKP) and hypervirulent Klebsiella pneumoniae (hvKP) and also provide an effective selection of drug in CRKP and hvKP treatment. Materials and methods Twelve strains were collected and investigated these isolates for their antimicrobial susceptibility and molecular features. Resistance mechanisms, virulence-associated genes, multilocus sequence typing (MLST), and serotypes were detected by PCR and sequencing. Next general sequencing (NGS) was carried out to determine the features of carbapenem resistance and virulence. The synergistic activity of tigecycline-imipenem (TGC+IPM), tigecycline-meropenem (TGC+MEM), and tigecycline-aztreonam (TGC+ATM) combinations were performed by microdilution checkerboard method. Results Eleven CRKP and one hvKP strains were collected. All strains showed highly sensitive rates to tigecycline (TGC) and amikacin (AMK). NDM (33.3%, 4/12) was the main resistance mechanism and MLST assigned 3 of them to ST11. CTX-M-producing (n = 1) and KPC-2-producing (n = 1) isolates belonged to ST147 and ST11, respectively. The MICs of ATM and quinolones in NDM-1 CRKP and NDM-5 CRKP strains were different. The serotype of the majority strains was KL22KL137 (58.3%, 7/12), hvKP stain belonged to K64. CRKP strains harbored plasmid-mediated quinolone resistance genes (oqxA, oqxB, qnrS, qnrB), β-lactams (bla CTX-M-3), aminoglycosides, type I and type III fimbriae genes, siderophore genes, and transporter and pumps. SIM-producing ST1764 K64 showed typical features of hvKP, showing hypermucoviscosity phenotype. The virulence genes, including rmpA2, alls and aerobactin genes, linked to hvKP, were found in ST1764 hvKP. hvKP was sensitive to quinolone; also, oqxA gene was detected. All TGC combinations showed highly synergistic effects and TGC+IPM was more effective treatment. Conclusion We first identified the NDM-5-producing ST690 CRKP and SIM-producing ST1764 hvKP strains in Shanxi province. Tigecycline-carbapenem combinations were available treatments for CRKP.

Food stuffs of animal origin may present hazards, due to bacterial contamination. This study was conducted to determine the prevalence, serotypes, and virulence genes of Escherichia coli in raw beef meat and some of meat products (raw, minced meat, beef burger and sausage, 25 sample each).Bacteriological examination of total 100 samples were collected from different localities in port-said City showed that 10/100 (10%) of samples were infected with E. coli. The prevalence of E.coli strains in fresh and minced meat, beef burger and sausage samples were4% (1/25), 16 % (4/25), 8 %( 2/25), and 12% (3/25), respectively.Serological typing were conducted on 10 isolates and results obtained revealed that two (20%) O114, 2 (20%) O157, three (30%) O125, and one (10%) for each of O127; O18, and O158. Ten isolates were submitted to molecular studies for detection of the traT (transfer protein), iutA (encoding aerobactin) and iss (increased serum survival protein) genes, by using PCR technique. PCR assays for different virulence genes showed that sex (60%), four (40%), and sex (60%) strains carry iss, iutA, and Trat genes, respectively.

Hypervirulent Klebsiella pneumoniae (hvKP) strains are associated with vigorous clinical presentation and relapses. Initially reported from Asia, these variants have spread globally and become an emerging agent of significant health threat. This study was carried out to identify hvKP strains in a previously uninvestigated region and to evaluate the impact of commonly-employed phenotypic and genotypic markers as diagnostic assays. A total of 111 blood culture isolates, collected at a tertiary care center was investigated. The hvKP strains were sought by a string test and the amplification of partial magA, rmpA, iucA and peg344. All products were characterized via sequencing. Evidence for hvKP was observed in 10.8% via iucA amplification (7.2%), string test (2.7%) and magA amplification (0.9%). Specific products were not produced by assays targeting rmpA and peg344 genes. Antibiotic susceptibility patterns compatible with possible extensive or pan-antimicrobial resistance was noted in 66.7% of the hvKP candidate strains. Capsule type in the magA positive strain was characterized as K5. We have detected hvKP in low prevalence at a region with no prior documentation. Targetting the aerobactin gene via iucA amplification provided the most accurate detection in this setting. The epidemiology of hvKP in Anatolia requires elucidation for effective control and management.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains have been increasingly reported, and it is important to understand the evolutionary mechanisms of these highly pathogenic and resistant bacterial pathogens. In this study, we characterized a ST11 carbapenem-resistant K. pneumoniae strain which harbored an IncFIB/IncHI1B type virulence plasmid and an IncFII/IncR type blaKPC–2-bearing plasmid. The virulence plasmid was found to be conjugative and harbored a 35-kbp fragment including aerobactin encoding cluster from virulence plasmid pLVPK and multiple resistance genes, resulting in a mosaic multi-drug resistance and virulence plasmid. This virulence plasmid could be transferred via conjugation to Escherichia coli and K. pneumoniae strains alone as well as together with the blaKPC–2-bearing plasmid. Co-transmission of virulence and blaKPC–2-bearing plasmids would directly convert a classic K. pneumoniae strain into CR-HvKP strain, leading to a sharp increase in the prevalence of CR-HvKP in clinical settings, which poses a great threat to human health.

The presence and dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) often cause life-threatening infections worldwide, but the therapeutic option is limited. In this study, whole-genome sequencing (WGS) was applied to assess the epidemiological characteristics and transmission dynamics of CRKP isolates recovered from two fetal outbreaks of nosocomial infections. Between April 2016 and March 2018, a total of 70 isolates of K. pneumoniae were collected from sterile samples in a tertiary hospital in Hangzhou, China. The minimal inhibitory concentrations (MICs) of 21 antimicrobial agents were determined using the broth microdilution methods. Pulsed-field gel electrophoresis (PFGE) was performed on 47 CRKP isolates, and 16 clonally related isolates were further characterized by Illumina sequencing. In addition, the complete genome sequences of three representative isolates (KP12, KP36, and KP37) were determined by Oxford Nanopore sequencing. The K. pneumoniae isolates were recovered from patients diagnosed with pulmonary infection, cancer, or encephalopathy. For all CRKP isolates, PFGE separated three clusters among all strains. The most predominant PFGE cluster contained 16 isolates collected from patients who shared close hospital units and represented a potential outbreak. All 16 isolates showed an extremely high resistance level (≥87.5%) to 18 antimicrobials tested but remain susceptible to colistin (CST). Multiple antimicrobial resistance and virulence determinants, such as the carbapenem resistance gene blaKPC-2, and genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA and rmpA2), were observed in the 16 CRKP isolates. These isolates belonged to sequence type 11 (ST11) and capsular serotype KL64. A core genome single nucleotide polymorphism (cgSNP)-based phylogenetic analysis indicated that the 16 CRKP isolates could be partitioned into two separate clades (≤15 SNPs), suggesting the two independent transmission scenarios co-occurred. Moreover, a high prevalence of IncFIB/IncHI1B type virulence plasmid with the iroBCDN locus deleted, and an IncFII/IncR type blaKPC-2-bearing plasmid was co-harbored in ST11-KL64 CRKP isolates. In conclusion, our data indicated that the nosocomial dissemination of ST11-KL64 CRKP clone is a potential threat to anti-infective therapy. The development of novel strategies for surveillance, diagnosis, and treatment of this high-risk CRKP clone is urgently needed.

IntroductionKlebsiella pneumoniae is recognized as an urgent threat to human health because of the emergence of multidrug-resistant (MDR) and hypervirulent strains. Development of novel highly effective and safe treatment options is the need of the hour. One of the ways of achieving this goal is by conducting molecular characterization studies of antibiotic resistant bacteria.Hypothesis/Gap StatementThe government of India is committed to generate validated AMR data from different parts of the country. Efforts are being made to perform molecular characterization of MDR and potentially virulent bacteria by whole-genome sequencing (WGS). However, the data that we have at present is skewed as many parts of the country remain underrepresented. Uttarakhand is one such state located in the Himalay an belt of India, with relatively poor access to healthcare and a paucity of research.AimThis study was performed to generate WGS based preliminary data about the population structure, multi-locus sequence types (MLST), and virulence factors of CRKp isolates recovered from patients in a tertiary care teaching hospital and institute of national importance, located in Rishikesh, Uttarakhand, India.MethodologyA cross-sectional study was conducted at a tertiary care teaching hospital. It included twenty-nine randomly selected and archived carbapenem resistant Klebsiella pneumoniae (CR-Kp) isolates obtained from various clinical samples submitted in the Bacteriology laboratory for culture and sensitivity testing, from July 2018 to August 2019. After preliminary identification (ID) and antibiotic susceptibility testing (AST), as per standard guidelines, these isolates were sent to Central Research Laboratory (CRL), Bengaluru, India, for further characterization & WGS.ResultsTwenty-seven out of twenty-nine test isolates were CRKp. Among the 27 CRKp isolates, ST14 was the most common sequence type (8, 29.6%), followed by ST231 (5, 18.5%) and ST147(3, 11.1%) respectively. KL2 (9/27, 33.3%) and KL51 (8/27, 29.6%) were dominant K loci types in this study. Out of 5 O antigens identified, O1 and O2 together accounted for 88.9% (n=27) CRKp isolates. Yersiniabactin and Aerobactin were identified in 88.9% (24/27) & 29.6% (8/27) of the CRKp isolates of the isolate. Regulatory genes rmpA2 and rmpADC were found in 14.8% (4/27) and 3.7% (1/27) isolates respectively. The predominant plasmid replicons present were ColKP3 (55.5%), IncFII(K) (51.8%), IncFIB(pQil) (44.4%), IncFIB(K) (37%), IncR (33.3%) and Col44 0I (18.5%) respectively. A perfect agreement (100%) was observed between phenotypic and genotypic resistance profiles in the case of fluoroquinolones, penicillins, Beta Lactam/Beta Lactam Inhibitor combinations (BL/BLI), and cephalosporins respectively. As compared to phenotypic resistance, higher genotypic resistance for aminoglycosides (96.3%) and folate pathway inhibitors (92.6%) respectively, was observed.ConclusionThis study emphasizes the need for continued genomic surveillance of emerging CRKp and other MDR bacteria in Uttarakhand and neighbouring states of India. This in turn would help in generating critical information that can be used to assess the emergence, dissemination, and potential impact of important variants.

The aim of this study was to examine antibiotic resistance profiles and diversity of β-lactamases in Escherichia coli present within the population and the potential spread of resistant E. coli into the receiving environment using city-scale sewage surveillance. In E. coli isolates from ECC plates without antibiotics from ten influent samples (n = 300), highest resistance was observed against ampicillin (16.6%), sulfamethoxazole (9.7%) and trimethoprim (9.0%), while in effluent samples (n = 262) it was against sulfamethoxazole (11.8%), ampicillin (11.5%) and tetracycline (8.8%). All isolates (n = 123) obtained on cefotaxime-containing plates were multidrug-resistant. Several clinically important antibiotic resistance genes (ARGs) were detected in 46 E. coli isolates subjected to whole-genome sequencing, including carbapenemases like NDM-6, VIM-1 and OXA-48-variant, as well as tigecycline resistance gene tet(X4). CTX-M-15 was the most prevalent (42.9%) extended-spectrum β-lactamase among cefotaxime-resistant isolates, followed by CTX-M-27 (31.4%) and CTX-M-14 (17.1%), resembling clinical prevalence in Norway. Most of the sequenced isolates carried other clinically relevant ARGs, such as dfrA17, sul1, sul2, tet(A), aph(6)-Id, aph(3’’)-Ib and aadA5. Sixteen different sequence types (STs) were identified, including ST131 (39.1%), ST38 (10.9%) and ST69 (8.7%). One E. coli isolate belonging to novel ST (ST11874) carried multiple virulence factors including genotoxin, salmochelin, aerobactin and yersiniabactin, suggesting that this isolate has potential to cause health concerns in future. Our study reveals presence of clinically relevant ARGs like blaNDM-6 and tet(X4) in pathogenic strains, which have so far not been reported from the clinics in Norway. Our study may thus, provide a framework for population-based surveillance of antibiotic resistance.

BackgroundHypervirulent Klebsiella pneumoniae (hvKp) infections have mainly been described in Asia. Two patients in the age group of 30 to 50 years presented within a two month period to a tertiary referral hospital in Texas with septic shock, hepatic abscess and septic thrombophlebitis. Blood cultures were positive for Klebsiella pneumoniae (isolates 2020CK-00441 and 2021CK-00720 respectively).MethodsWhole genome sequencing was performed using paired-end Illumina MiSeq reads for both isolates. Nanopore sequencing to obtain a closed genome was performed for 2020CK-00441.Results2020CK-00441 belonged to ST23 type while 2021CK-00720 was a ST65 type isolate. Kleborate analyses predicted with high confidence that both the isolates were hvKp. Phylogenetic analyses showed that the two strains are not closely related to each other or to any other known hvKp isolates. Both the isolates had yersiniabactin, colibactin, aerobactin and salmochelin producing loci which likely confer these isolates hvKp phenotype. 2020CK-00441 had a unique pK2044 like plasmid.ConclusionsHvKp strains capable of causing devastating metastatic septic infections have emerged in Texas. These isolates are unique when compared to other hvKp strains of the world. Country wide surveillance and whole genome sequencing of these strains is essential to prevent a major public health emergency in USA.

Background Two different types of hypervirulent K. pneumoniae (HvKp), the MLST-11 and serotype K1/K2 strains, have been frequently described in recent studies. Although these two types of strains were described to be HvKp, their virulence was not compared. In this study, in vitro and in vivo approaches were used to assess differences in virulence. Materials and methods A total of twenty-nine isolates, including 6 strains of each of serotype K1 and K2 isolates and 17 strains of ST11 isolates, were selected for this study. Phenotypic tests of virulence were performed by the string test and analysis of the virulent associated genes was detected by PCR. In vitro models of serum resistance and phagocytosis were used as the parameters to assess the virulence. In-frame deletion of virulence-associated genes was performed to study their contributions to virulence. The median lethal dose, i.e., the LD50, in mice was determined following IP injection. Results Although serotype K1 and K2 strains and ST11 isolates had similar virulence gene profiles, the ST11 isolates showed less serum and phagocytic resistance than the serotype K1/K2 isolates. The mouse lethality test revealed that all ST11 isolates were unable to cause lethality, even at > 107 CFU, while serotypes K1 and K2 showed an LD50 at ≤ 103 CFU. Aerobactin or capsule knockout mutants exhibited a lower LD50 than the parental strain, while capsule mutants showed a more significant decrease in LD50. Conclusion Since there was a significant difference in virulence levels between the two types of HvKp when assessed in in vitro and in vivo models, it may be better to use the designation "HvKp" for some strains based on animal studies to avoid confusion. Virulence and non-virulence could be analysed in a relative manner, especially in comparison studies.

We researched 26 K. pneumoniae ssp. pneumoniae strains, isolated from patients and objects of the environment during the epidemic ill-being period in the pediatric hospital. Identification of strains and their clustering, study of antibiotic sensitivity, detection of antibiotic resistance determinants, whole genome sequencing were carried out. It is established that one strain characterized by identical RAPD patterns and sensitivity to antibiotics from the groups of cephalosporins, monobactams, aminoglycosides, carbapenems, fluoroquinolones, sulfonamides and glycylcyclides circulates in the department.The strain is resistant to ampicillin (MIC > 16) and nitrofurantoin (MIC > 64). Whole genome sequencing made it possible to characterize the strain as K. pneumoniae ssp. pneumoniae ST 3181K-54, the analysis of the whole genome sequence showed the presence of a fosomycin resistance gene (fosA) and virulence determinants namely a complex of siderophores (aerobactin and enterobactin), fimbria of the third type, iron utilization system kfu and cluster of genes responsible for allantoin utilization that allowed us to consider the strain as a microorganism with a high pathogenic potential capable to rapid prevalence and dissemination in tissues.

Objectives To investigate the pathogenicity of Klebsiella pneumoniae (KPN) possessing rmpA or the aerobactin gene in infected mice. Methods BALB/c mice were divided into four groups (n = 10 per group) and infected with: string test-positive and rmpA-positive or aerobactin-positive KPN (group 1), string test-negative but rmpA-positive KPN (group 2), string test-negative but aerobactin-positive KPN (group 3), or string test- and rmpA/aerobactin-negative KPN (group 4). Mouse survival time was compared among groups, and the infection of livers, spleens, lungs, and kidneys and KPN growth were assessed in infected mice. Results Compared with the negative group (group 4), the survival rates of mice infected with rmpA- or aerobactin-positive KPN (groups 1–3) were significantly lower, their multi-organ injuries were significantly more severe, and bacterial enumeration was significantly higher. Conclusions Despite being string test-negative, aerobactin- or rmpA-positive KPN still exhibit high virulence and anti-immune effect activity. Therefore, the combination of the string test and gene detection of aerobactin and rmpA will be helpful in screening hypervirulent KPN.

Thirty-five serotype K1 hypervirulent Klebsiella pneumoniae (K1-hvKP) isolates collected from a Chinese hospital during the whole year of 2017 were evaluated to characterize the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes. In total, 18 (51.4%) isolates were detected to carry PMQR genes, and the most frequently detected gene was qnrS1 (37.5%), followed by aac(6')-Ib-cr (15%) and qnrB4 (2.5%). Remarkably, some qnr-carrying strains had only a single or more quinolone resistance-determining region mutations in GyrA or ParC, and most exhibited high-level ciprofloxacin resistance. However, only 11.4% (4/35) isolates were resistant to quinolones. Furthermore, 34.3% (12/35) carried extended-spectrum β-lactamase (ESBL) genes, but only 14.3% (5/35) exhibited ESBL phenotype. The most prevalent virulence genes were mrkD (100%, 21/21), followed by magA (97.1%, 34/35), wabG (97.1%, 34/35), rmpA (97.1%, 34/35), aerobactin (94.3%, 33/35), kfuB (94.3%, 33/35), ycf (91.4%, 32/35), iutA (91.4%, 32/35), rmpA2 (62.9%, 22/35), wcaG (62.9%, 22/35), ybtS (51.4%, 18/35), allS (45.7%, 16/35), and iroN (22.9%, 8/35). Multilocus sequence typing (MLST) analysis assigned the 35 K1-hvKP isolates into 5 sequence types (STs), with ST23 encompassing 77.1% of the strains. Pulsed field gel electrophoresis (PFGE) typing showed that strains closely related by MLST clustered in major PFGE clusters, of which cluster A accounts for 16 ST23 isolates and cluster B includes 11 ST23 isolates. The analysis of the transconjugants showed decreased susceptibility to quinolones and revealed a cotransfer of blaCTX-M-15 with the qnrS1 alleles. Cumulatively, our findings showed that the PMQR-producing K1-hvKP strain is covertly spreading even when K1-hvKP is rarely resistant to fluoroquinolones (FQs) according to the Clinical and Laboratory Standards Institute criteria. It is therefore critical to continuously monitor the PMQR-producing K1-hvKP epidemiology and minimize potential risks from FQ-resistant K1-hvKP.

Objectives The relevance of Escherichia coli associated bacteriuria infection in pregnant women is poorly understood, despite these strains sharing a similar virulence profile with other pathogenic E. coli causing severe obstetric and neonatal infections. We characterized and determined the antimicrobial susceptibility, resistance genes and virulence profiles of 82 E. coli isolates associated with asymptomatic bacteriuria in some pregnant in Ghana from February to August 2016 using Kirby–Bauer disc diffusion and polymerase chain reaction. Results High levels of antimicrobial resistance were observed to ampicillin (79.3%), tetracycline (70.7%) and cotrimoxazole (59.8%), except for cefuroxime (32.9%). Resistance genes analyses revealed 58.5% were positive for Bla TEM and 7.3% for aph(3)-Ia(aphA2). Virulence factors (VFs) was more widespread in pregnant women in the 2nd and 3rd trimesters than 1st trimester. VFs relating to adhesion (papC and iha), Protectins (traT), aerobactin acquisition (iutA) and iron acquisition systems (fyuA and irp2) were more prevalent in the resistant E. coli isolates. This study provides evidence for a link in bacteriuria and transmission of extra-intestinal E. coli in pregnant women to cause multi-resistant obstetric or neonatal infections. Considering the involvement of extra-intestinal E. coli in infections, results are helpful to develop strategies to prevent maternal and/ neonatal infections.

Jiaying Tan,1,* Mi Tian,1,* Feng Zhao,1 Shuixiang Deng,1 Peng Jin,1 Yao Wang,1 Huimei Wen,1 Xiaohua Qin,2,3 Ye Gong1,4 1Department of Critical Care Medicine, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China; 2Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China; 3Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, 200040, People’s Republic of China; 4Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaohua QinInstitute of Antibiotics, Huashan Hospital, Fudan University, No. 12 Middle Urumqi Road, Shanghai, 200040, People’s Republic of ChinaEmail 0456240@fudan.edu.cnYe GongDepartment of Critical Care Medicine, Huashan Hospital, Fudan University, No. 12 Middle Urumqi Road, Shanghai, 200040, People’s Republic of ChinaEmail gong_ye@fudan.edu.cnBackground: Male genitourinary abscess is one of the serious complications of urinary tract infections (UTIs). There were few researches on the clinical and pathogenic characteristics of male genitourinary abscess.Patients and Methods: A retrospective observational study was conducted between January 2004 and April 2019. Male patients with genitourinary abscess originated from urinary tract, including sites of scrotum, testis, epididymis, spermatic cord, and prostate, were enrolled. Clinical and microbial records were collected and analyzed, and antimicrobial susceptibility testings wereperformed according to CLSI standard. Whole-genome sequencing was applied to detect the β-lactamase genes and virulence genes, as well as to determine the multilocus-sequence typing (MLST) of the collected Klebsiella pneumoniae (K. pneumoniae) isolates.Results: A total of 22 male patients were included. The main clinical symptoms were fever (86.4%), scrotal swelling (68.2%), local skin warmth (59.1%) and ulceration (45.5%). Urinary irritation was often presented in prostate involved abscess. Ultrasound features had a 94.7% positive rate. Surgical treatment, including abscess drainage, was helpful to the prognosis. No matter where the specimens obtained from, including blood, urine or pus, multidrug-resistant K. pneumoniae was the dominant (11 cases, 50.0%) microorganism in positive cultures. Nine of eleven K. pneumoniae isolates had been preserved and recovered. As for MLST typing, all the nine available isolates of K. pneumoniae belonged to the ST11 type and characterized with blaKPC-2 carbapenemase gene. Virulence genes rmpA2, ybtS, kfuC, wzi, aerobactin genes (iucABCD and iutA) and type 3 fimbriae genes (mrkAD) were identified in all the K. pneumoniae isolates.Conclusion: It seemed that more patients under 35 years old were vulnerable to genitourinary abscess. There was an increasing trend that multidrug-resistant K. pneumoniae isolates with multiple virulence genes were involved in male genitourinary abscess. Prompt and proper antibiotic use, combined with adequate drainage of the abscess, was important to prognosis.Keywords: male genitourinary abscess, carbapenem-resistant Klebsiella pneumoniae, virulence genes, MLST type

The increase in infections with multidrug-resistant and virulent Klebsiella pneumoniae (K. pneumoniae) strains poses a serious threat to public health. However, environmental reservoirs and routes of transmission for Klebsiella spp. that cause infections in humans and in livestock animals are not well understood. In this study, we aimed to analyze the distribution of antibiotic resistance genes and important virulence determinants (ybt, clb, iro, iuc, rmpA/A2) among 94 Klebsiella spp. isolates from different animal and food sources isolated between 2013 and 2017 in Germany. Antibiotic susceptibility testing was performed, and the genomes were sequenced by Illumina and Nanopore technology. Genetic relationships were assessed by conducting core genome multilocus sequence typing (cgMLST). Kleborate was used to predict resistance and virulence genes, Kaptive was used to derive the capsule types. The results revealed that 72 isolates (76.6%) belonged to the K. pneumoniae sensu lato complex. Within this complex, 44 known sequence types (STs), 18 new STs, and 38 capsule types were identified. Extended-spectrum beta-lactamase (ESBL) genes were detected in 16 isolates (17.0%) and colistin resistance in one (1.1%) K. pneumoniae isolate. Virulence genes were found in 22 K. pneumoniae isolates. Overall, nine (9.6%) and 18 (19.1%) isolates possessed the genes ybt and iuc, respectively. Notably, aerobactin (iuc lineage 3) was only detected in K. pneumoniae isolates from domestic pigs and wild boars. This study provides a snapshot of the genetic diversity of Klebsiella spp. in animals and food products in Germany. The siderophore aerobactin was found to be more prevalent in K. pneumoniae strains isolated from pigs than other sources. Further investigations are needed to evaluate if pigs constitute a reservoir for iuc lineage 3.

We describe a case of Lemierre's syndrome (LS) caused by a hypervirulent strain of Klebsiella pneumoniae in a 63-year-old female with hypertension, hyperlipidemia, and diabetes mellitus, who presented with right neck pain and fevers. Computerized tomography of the neck and chest revealed an occluded right internal jugular vein secondary to thrombosis and septic emboli in lungs. Blood cultures grew K. pneumoniae. The patient was treated with ampicillin-sulbactam and then transitioned to amoxicillin-clavulanate to complete a 6-week course of antibiotics, and a 3-month course of rivaroxaban. String test of the K. pneumoniae isolate was positive at 2 cm. Whole genome sequencing identified several genes associated with the hypervirulent strain, notably the genes encoding for aerobactin (iucA and iucB) and salmochelin (iroB) iron acquisition systems. LS can rarely be caused by K. pneumoniae. Clinicians should monitor for known complications, such as septic emboli in patients with LS.

Wastewater treatment plants (WWTPs) are significant reservoirs of bacterial resistance. This work aims to identify the determinants of resistance produced by Gram-negative bacteria in the influent and effluent of two WWTPs in Portugal. A total of 96 wastewater samples were obtained between 2016 and 2019. The numbers of total aerobic and fecal contamination bacteria were evaluated, and genomic features were searched by polymerase chain reaction (PCR) and Next-Generation Sequencing (NGS). Enterobacteriaceae corresponded to 78.6% (n = 161) of the 205 isolates identified by 16sRNA. The most frequent isolates were Escherichia spp. (57.1%, n = 117), followed by Aeromonas spp. (16.1%, n = 33) and Klebsiella spp. (12.7%, n = 26). The remaining 29 isolates (14.1%) were distributed across 10 different genera. Among the 183 resistant genes detected, 54 isolates produced extended spectrum β-lactamases (ESBL), of which blaCTX-M-15 was predominant (37 isolates, 68.5%). A KPC-3 carbapenemase-producing K. oxytoca was identified (n = 1), with blaKPC-3 included in a transposon Tn4401 isoform b. A higher number of virulence genes (VG) (19 genes) was found in the E. coli 5301 (O25b-ST131-B2) isolate compared with a commensal E. coli 5281 (O25b-ST410-A) (six genes). Both shared five VG [Enterobactin, Aerobactin, CFA/1 (clade α), Type1 (clade γ1), Type IV]. In conclusion, this work highlights the role of relevant clinical bacteria in WWTPs, such as KPC-3-producing K. oxytoca, and, for the first time, a CTX-M-15-producing Ochromobactrum intermedium, a human opportunistic pathogen, and a SED-1-producing Citrobacter farmeri, an uncommon CTX-M-type extended-spectrum beta-lactamase.

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant K. pneumoniae are on the rise globally. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in present study. K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were subjected to 96-well plate biofilm formation assay followed by MBEC assay for subset of strong biofilm formers. Whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure, hence MICs cannot be extrapolated for treatment. The biofilm forming ability had a significant association with morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (p < 0.05) resulted in fewer “average days alive” for the patient (3.33 days) in comparison to those negative for biofilms (11.33 days). Phylogenetic analysis including global isolates revealed clear association of sequence types with genes for biofilm formation and carbapenem resistance. Known hypervirulent clone-ST23 with wcaG, magA, rmpA, rmpA2, and wzc with lack of mutation for hyper-capsulation might be poor biofilm formers. ST15, ST16, ST307, and ST258 (reported global high-risk clones) were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, and mrkD) and pili (pilQ and ecpA). This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.

Escherichia coli is one of the most commonly isolated pathogens from surgical site infections (SSIs) that accounts for significant morbidity and mortality, especially with high virulent strains. The purpose of this study was to evaluate the virulence determinants of E. coli strains isolated from surgical sections. 51 E. coli isolates were assessed for biofilm formation, mannose-sensitive or -resistant hemagglutination, capsule, hemolysin and siderophores production. Antibiotic susceptibility against 14 common antibiotics was performed. As a result, 25 strains displayed 5 virulence factors and multiple drug resistance towards more than 11 antibiotics. DNA and plasmids were extracted from the 25 virulent isolates. PCR was used to investigate virulence genes on DNA encoding adhesins (fimH-1, mrkD), hemolysin (hlyA), siderophores (entB: enterobactin, iutA: aerobactin, irp-1: yersiniabactin- which was encoded on (HPI) high-pathogenicity island). MrkD, iutA and hlyA genes were also screened on extracted plasmids. The most common virulence genes were iutA (25/100%), mrkD (24/96%), entB (23/92%) and fimH-1 (21/84%). Irp1 was found at moderate rates (15 /60%) and at lower prevalence, was gene hlyA (2 / 8%). Plasmids were found in 16/25 strains. MrkD and iutA were present in 10 /16 plasmids, whereas none of them harbored hlyA. In conclusion, most E. coli isolates harbored high frequencies of (fimH1, mrkD, entB and iutA) which seem to be at the basis of pathogenicity. However, some strains, which carry HPI and have virulence plasmids can account for even more real threat if they spread among other Enterobacteriaceae members in surgical sections.

Klebsiella pneumoniae (Kp) reference strain Kp52.145 is widely used in experimental Klebsiella pathophysiology. Since 1935, only one other strain of the same sublineage (sequence type ST66, capsular serotype K2) was isolated (AJ210, Australia). Here, we describe a community-acquired invasive infection caused by a ST66-K2 Kp strain in France. Four hypermucoviscous Kp isolates responsible for acute otitis media, meningitis, bacteraemia and bacteriuria, respectively, were obtained from a patient with a history of chronic alcoholism and diabetes mellitus, and infected with HIV. The isolates were characterized by phenotypic and genomic methods. The four genetically identical ST66-K2 isolates presented a full antimicrobial susceptibility profile, including to ampicillin, corresponding to a single strain (SB5881), which was more closely related to AJ210 (135 SNPs) than to Kp52.145 (388 SNPs). Colibactin and yersiniabactin gene clusters were present on the integrative and conjugative element ICEKp10 in the chromosome. The two plasmids from Kp52.145 were detected in SB5881. In addition to carrying genes for virulence factors RmpA, aerobactin and salmochelin, plasmid II has acquired in SB5881, the conjugation machinery gene cluster from plasmid I. We report the first case of community-acquired infection caused by a hypervirulent ST66-K2 Kp strain in Europe. This demonstrates the long-term persistence of the high-virulence and laboratory model ST66-K2 sublineage. The combination of a conjugative apparatus and major virulence genes on a single plasmid may contribute to the co-occurrence of hypervirulence and multidrug resistance in single Kp strains.

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant (CR) K. pneumoniae are on the rise in India. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in the present study.K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were tested for antimicrobial susceptibility as recommended by CLSI 2019 and subjected to 96-well microtitre plate biofilm formation assay. Based on optical density at 570 nm isolates were graded as strong, moderate and weak biofilm formers. Subset of strong biofilm formers were subjected to whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Biofilm formation was compared for an association with the carbapenem susceptibility and with patient outcome. Statistical significance, correlations and graphical representation were performed using SPSS v23.0.Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure. The biofilm forming ability had a significant association with the morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (PwcaG, magA, rmpA, rmpA2 and wzc with a lack of mutation for hyper-capsulation might be poor biofilm formers. Interestingly, ST15, ST16, ST307 and ST258 – reported global high-risk clones were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, mrkD) and pili (pilQ, ecpA).This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and antimicrobial resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.

Background and Objectives: Escherichia coli is known to be the pathogen commonly isolated from those infected with uri- nary tract infections (UTIs). The aim of this study was to investigate the presence of E. coli virulence genes and antibiotics’ resistance pattern among clinical isolates in the Northeast of Iran. Relationships between virulence genes and antimicrobial resistances were studied as well. Materials and Methods: Three hundred isolates of E. coli were isolated from patients with UTIs that referred to Ghaem and Imam Reza hospitals (Mashhad, Iran) during August 2016 to February 2017. A multiplex PCR was employed to amplify the genes encoding pyelonephritis associated pili (pap), S-family adhesions (sfa), type1fimbriae (fimH) and aerobactin (aer). Disk diffusion test was performed to test the susceptibility of isolates to β-lactams, aminoglycosides, cephalosporins, quino- lone, fluoroquinolones, carbapenems and trimethoprim-sulfamethoxazole. Results: The PCR results identified the fimH in 78.4%, aer in 70.5%, sfa in 13.6% and the pap in 8.2% of isolates. The rates of antibiotic resistance of the isolates were as follows: 64.7% resistant to cephalosporins, 34% to trimethoprim-sul- famethoxazole, 31% to fluoroquinolones, 15.3% to aminoglycosides, 13.3% to β-lactams, 7.8% to quinolones and 4.4% to carbapenems. Significant relationships existed between pap and aer, pap and sfa, aer and fluoroquinolones also pap and cephalosporins. Conclusion: fimH and aer were found in > 50% of isolates suggesting the importance of both genes in UPEC. The majority of isolates had fimH as adhesion factor for colonization. Determining antibiotic resistance patterns in specific geographical areas is necessary for appropriate treatment of urinary tract infection. The high rate of resistance to cephalosporins is most likely due to incorrect drug administration

Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes., Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin.

Multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKP) has been increasingly reported and is now recognized as a significant threat to public health; however, characterization of MDR-hvKP has not been systematically investigated. In the present study, 124 of 428 (28.92%) K. pneumoniae clinical isolates collected from January 2010 to December 2016 were identified with aerobactin and defined as hvKP; these included 79 (48.77%) non-MDR-KP, 35 (15.63%) extended spectrum β-lactamase-producing K. pneumoniae (ESBL-KP), and 10 (23.81%) carbapenem-resistant K. pneumoniae (CR-KP) isolates. The remaining 304 isolates that were not hypervirulent phenotypes were defined as classic K. pneumoniae (cKP). The antimicrobial resistance rate of cKP was significantly higher than that of the hvKP isolates in the non-MDR-KP group, but showed no significant differences in the ESBL-KP and CR-KP groups. The detection frequencies of K1 serotype, hypermucoviscosity, sequence type 23 (ST23), and two virulence genes—mucoviscosity-associated gene A (magA) and regulator of mucoid phenotype A (rmpA)—were significantly high in the hvKP than cKP isolates in all three groups (P < 0.05). Most of the hypervirulent ESBL-KP and CR-KP isolates were K non-typeable (28/45) and harbored at least one gene for virulence (36/45). The hypervirulent ESBL-KP isolates primarily carried blaSHV (25/35, 71.42%) genes, and the hypervirulent CR-KP isolates mainly carried blaNDM-1 (8/10, 80%) genes. Moreover, four hypervirulent ESBL-KP and two hypervirulent CR-KP isolates showed resistance to tigecycline. The transcriptional levels of rmpA in cKP were much lower than that in hvKP isolates in all three groups. Furthermore, overexpression of rmpA could partly restore the hypervirulent phenotype of the rmpA-low-expression cKP isolates. In conclusion, our data suggest that the K1 serotype, rmpA, magA, hypermucoviscosity, and ST23 were strongly associated with hvKP in non-MDR-KP, ESBL-KP, and CR-KP groups, and low rmpA expression levels contributed to the absence of hypervirulent phenotype.

Background Klebsiella pneumoniae is a leading cause of bloodstream infection (BSI). Strains producing extended-spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial-resistant (AMR) K. pneumoniae and also for the characteristically antimicrobial-sensitive, community-acquired “hypervirulent” strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide. Methods We conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate, a K. pneumoniae-specific genomic typing tool. Results K. pneumoniae BSI isolates were highly diverse, comprising 120 multi-locus sequence types (STs) and 63 K-loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR-virulence convergence, which is generally considered a rare phenomenon but was particularly common among South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co-transfer of these phenotypes among K. pneumoniae. Conclusions K. pneumoniae BSI in South and Southeast Asia are caused by different STs from those predominating in other regions, and with higher frequency of acquired virulence determinants. K. pneumoniae carrying both iuc and AMR genes were also detected at higher rates than have been reported elsewhere. The study demonstrates how genomics-based surveillance—reporting full molecular profiles including STs, AMR, virulence and serotype locus information—can help standardise comparisons between sites and identify regional differences in pathogen populations.

Aim. Klebsiella pneumoniae is one of the most important causes of nosocomial infections, including pneumonia, sepsis, and urinary tract infection. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique is a quick, reliable, and cost-effective method for molecular typing of Enterobacteriaceae family members. This study aimed to detect genetic relatedness among K. pneumoniae isolates from hospitals in Hamadan city, using ERIC-PCR technique. Materials and Methods. A total of 72 K. pneumoniae isolates were collected from patients admitted to Besat and Sina hospitals. After detection and confirmation of K. pneumonia isolates by chemical and conventional microbiological methods, DNAs were extracted after 24 hours of incubation at 37°C, using the boiling method. ERIC-PCR technique was carried out, and the ERIC patterns were analyzed by online data analysis service (inslico.ehu.es). ERIC profiles were compared using Dice method and clustered by UPGMA (unweighted pair group method with arithmetic mean) program. Also, the samples were evaluated by PCR method for the detection of aerobactin gene within their genome. Finding. The genetic relatedness among K. pneumoniae isolates was studied, and results established the genetic diversity of the clinical isolates by detecting 25 different ERIC types, including 14 common types and 11 unique types. Also, none of the isolates had aerobactin gene. Discussion. The results of this study showed high genetic diversity among K. pneumoniae strains, indicating the polyclonal distribution of K. pneumoniae isolates in Hamadan hospitals. This diversity causes problems for the treatment of infections due to the circulation of diverse K. pneumoniae clones, which possibly have different antimicrobial susceptibility patterns.

Multidrug-resistantKlebsiella pneumoniaeand hypervirulentK. pneumoniae(hvKP) have traditionally been considered two individual populations; however, strains displaying both phenotypes have emerged during the recent decade. Understanding the genotypic and phenotypic basis of the convergence could be of clinical importance. In this study, we aimed to evaluate the pathogenicity associated with different combinations of genotypes (i.e., sequence types, virulence factors, and capsular serotypes) and phenotypes (i.e., hypermucoviscosity and drug susceptibility) inK. pneumoniae.A total of 11K. pneumoniaeisolates causing bloodstream infections were included in the study, and they were assigned to seven STs (ST23, ST15, ST268, ST660, ST86, ST65, and ST1660) and carried various K-loci (KL1, KL2, KL16, KL20, and KL24). Hypermucoviscosity was observed for six isolates.bla(KPC-2)was detected in six carbapenem-resistant isolates, and the remaining ones were either multidrug-resistant or resistant to two types of antibiotics. Aerobactin- and yersiniabactin-encoding genes were detected in all isolates. AlthoughrmpA2was detected in all isolates, most contained frameshift mutations (82%). Genes encoding salmochelin, RmpA, and PEG344 were detected in seven isolates. Colibactin-encoding genes were carried by six isolates. Discrepancies among measured virulence inGalleria mellonellaand the serum-killing assay, and genotypes and phenotypes were detected. The results illustrate the complexity and difficulty with the current knowledge of hypervirulence to predict the phenotype by using genetic and phenotypic markers. Additionally, the emergence of carbapenem resistance in two isolates of KPC-2-producing hvKP of different sequence types emphasizes the urgency with which reliable clinical diagnostics for hvKP is needed. Funding Agencies|National Key Research and Development Program of China [2017YFC1200200]; Major Infectious Diseases such as AIDS and Viral Hepatitis Prevention and Control Technology Major Projects [2018ZX10712-001]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China [81702045, 81361138021]; Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas)Swedish Research Council Formas [2016-00640]; Swedish Foundation for International Cooperation in Research and Higher Education (STINT) [CH2016-6707]; Shenzhen Basic Research Projects [JCYJ20190807144409307]

Background The way of treating different types of infectious diseases is really important. Using genotyping method, we can determine the genetic relatedness between the organisms with different resistance profile from different sources. The aim of this study was to determine antibiotic resistance and genotyping of uropathogenic Escherichia coli (UPEC) strains using pulsed field gel electrophoresis (PFGE). Method Escherichia coli (E. coli) strains were recovered from the patients with urinary tract infections (UTI) whom admitted in several major hospitals in Tehran. Antibiotic susceptibility testing was done according to CLSI guideline. The present of some virulence factor have been detected using PCR assay. Genotyping of the strains was performed by PFGE and all PFGE profiles were subjected to data processing. Result In total, 60 E. coli strains were subjected to the study. Most of E. coli isolates were resistant to cefepime (100%) and cephalothin (74%) and susceptible to imipenem (100%), vancomycin (100%) and doxycycline (100%). Among the UPEC isolates the prevalence of fimbriae type I (fimH), hemolysin (hlyA) and aerobactin (aer) genes were 89%, 60% and 90%, respectively. The PFGE differentiated E. coli strains into 33 different genetic clusters. Majority (30%) of them including PFGE type 11 generated 15 bands, while PFGE type 2 was the lowest (2%) prevalent group with 9 bands. Conclusion The result showed that the antibiotic resistance is escalating rapidly. UPEC strains causing infections are more likely to harbor certain virulence genes. Our finding also showed E. coli strains isolated under the study were belonged to the diverse clones.

Background Aerobactin is a critical factor for the hypervirulent Klebsiella pneumoniae (hvKp), but data for the aerobactin-positive genotype of hvKp in elderly persons with ventilator-associated pneumonia (VAP) is limited. The purpose of this study is to understand the risk factors and characteristics of the hvKp genotype for elderly patients with VAP. Methods A retrospective study of 73 elderly patients with Kp was conducted from November 2008 to December 2017 in two tertiary hospitals. The clinical and microbiological data, including inflammatory reaction, nutritional status, antimicrobial susceptibility testing, string test, extended-spectrum-β-lactamase (ESBL) production, virulence-associated gene (capsular serotype-specific gene and rmpA/A2,magA,aerobaction) and multilocus sequence typing, of the hvKp group defined as aerobactin positive were compared with those of classic Kp strains. Results Of 73 Kp isolates, 46.6% were hvKp. ST23 is highly prevalent in two hospitals but is not highly associated with hvKp in different hospitals. Additionally, ST23, ST37 and ST2906 are more likely to induce lethal VAP. Most hvKp strains are sensitive to common antibiotics, but the number of multidrug-resistant (MDR) hvKp is increasing. Importantly, 38.2% of hvKp isolates produced ESBLs. Hypermucoviscosity and virulence-associated genes (K1,magA and rmpA/A2) were highly clustered in the hvKp group (P

Background The degree to which the chromosomal mediated iron acquisition system contributes to virulence of many bacterial pathogens is well defined. However, the functional roles of plasmid encoded iron acquisition systems, specifically Sit and aerobactin, have yet to be determined for Salmonella spp. In a recent study, Salmonella enterica strains isolated from different food sources were sequenced on the Illumina MiSeq platform and found to harbor the incompatibility group (Inc) FIB plasmid. In this study, we examined sequence diversity and the contribution of factors encoded on the IncFIB plasmid to the virulence of S. enterica. Results Whole genome sequences of seven S. enterica isolates were compared to genomes of serovars of S. enterica isolated from food, animal, and human sources. SeqSero analysis predicted that six strains were serovar Typhimurium and one was Heidelberg. Among the S. Typhimurium strains, single nucleotide polymorphism (SNP)-based phylogenetic analyses revealed that five of the isolates clustered as a single monophyletic S. Typhimurium subclade, while one of the other strains branched with S. Typhimurium from a bovine source. DNA sequence based phylogenetic diversity analyses showed that the IncFIB plasmid-encoded Sit and aerobactin iron acquisition systems are conserved among bacterial species including S. enterica. The IncFIB plasmid was transferred to an IncFIB plasmid deficient strain of S. enterica by conjugation. The transconjugant SE819::IncFIB persisted in human intestinal epithelial (Caco-2) cells at a higher rate than the recipient SE819. Genes of the Sit and aerobactin operons in the IncFIB plasmid were differentially expressed in iron-rich and iron-depleted growth media. Conclusions Minimal sequence diversity was detected in the Sit and aerobactin operons in the IncFIB plasmids present among different bacterial species, including foodborne Salmonella strains. IncFIB plasmid encoded factors play a role during infection under low-iron conditions in host cells. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3954-5) contains supplementary material, which is available to authorized users.

Objective: Escherichia coli is commensal bacterium of human intestine. The gut is a common pool of E. coli isolates causing urinary tract infections (UTIs). Some of fecal E. coli (FeEC) by the possession of certain virulence factors is able to cause diseases in human and other mammalian models. To evaluate the health threats coordinated with a given fecal source of E. coli strains, we determined the frequency of genes expressing virulence determinants in fecal E. coli isolates collected from human feces in Zabol, southeast of Iran. Methods: Escherichia coli isolates (n = 94) were separated from the feces of patients attending teaching hospitals, and screened for various virulence genes: fimH, his, hlyA, ompT, irp2, iucD, iroN, and cnf1 by using the multiplex polymerase chain reaction (PCR) method. Results: The prevalence of virulence genes was as follows: adhesins (fimH, 98% and iha, 26%), alpha-hemolysins (hlyA, 10%), outer membrane protease (ompT, 67%), aerobactin (iucD, 67%), iron-repressible protein (irp2, 91%) and salmochelin (iroN, 33%) and cytotoxic necrotizing factor 1 (cnf1). According to the diversity of different virulence genes, the examined isolates exhibited 29 different patterns. Conclusion: Our results demonstrated that most of the assessed isolates harbored several virulence factors. Our findings propose possibility of human feces serving as a source for pathogenic organisms, supporting the notion that fecal materials of humans play a role in the epidemiological chain of extra-intestinal pathogenic E. coli. This is the first report of the frequency of virulence factors among E. coli isolates collected from human feces in Iran.

Background The neonatal meningitis E. coli (NMEC) strain S88 carries a ColV plasmid named pS88 which is involved in meningeal virulence. Transcriptional analysis of pS88 in human serum revealed a strong upregulation of an ORF of unknown function: shiF, which is adjacent to the operon encoding the siderophore aerobactin. The aim of this work is to investigate the role of shiF in aerobactin production in strain S88. Results Study of the prevalence of shiF and aerobactin operon in a collection of 100 extra-intestinal pathogenic E. coli strains (ExPEC) and 50 whole genome-sequenced E. coli strains revealed the colocalization of these two genes for 98% of the aerobactin positive strains. We used Datsenko and Wanner’s method to delete shiF in two S88 mutants. A cross-feeding assay showed that these mutants were able to excrete aerobactin meaning that shiF is dispensable for aerobactin excretion. Our growth assays revealed that the shiF-deleted mutants grew significantly slower than the wild-type strain S88 in iron-depleted medium with a decrease of maximum growth rates of 23 and 28% (p shiF deleted mutants after growth in iron-depleted medium and found that these mutants secreted significantly less aerobactin than S88 (− 52% and - 49%, p Conclusions ShiF is physically and functionally linked to aerobactin. It provides an advantage to E. coli S88 under iron-limiting conditions by increasing aerobactin secretion and may thus act as an auxiliary virulence factor.

Background The definition of hypervirulent Klebsiella pneumoniae (hvKp), traditionally regarded as hypermucoviscosity, is controversial. However, data based on both phenotype (hypermucoviscous) and genetic (aerobactin) criteria are limited. Methods A retrospective study was conducted in 175 geriatric patients between January 2008 and January 2014. The clinical and molecular data, including antimicrobial susceptibility testing, extended-spectrum-β-lactamase (ESBL) production, virulence gene, and multilocus sequence typing of the hvKp-group (hypermucoviscosity and aerobactin positive) were compared with those of classic K. pneumoniae (cKp) isolates. Results Of 175 Kp isolates, 45.7% were hvKp. In pathogenicity, K1, K2, magA, rmpA, and rmpA2 genes were strongly associated with hvKp (P

AIMS In this study, the association between multidrug resistance (MDR) and the expression of some virulence factors were evaluated in Escherichia coli strains isolated from infant faeces and fresh green vegetables. The effect of isolate origin on associated virulence factors was evaluated. In addition, genetic fingerprinting of a sample of these isolates (10 isolates from each group) was studied in order to detect any genetic relatedness among these isolates. METHODS AND RESULTS Escherichia coli isolates were divided into four groups based on their origin (human faeces or plant) and their antibiotic resistance (multiresistance or susceptible). PCR was used to investigate heat-labile and heat-stable enterotoxin genes, and four siderophore genes (aerobactin, enterobactin, salmochelin and yersiniabactin). Genetic fingerprinting of the isolates was performed using enterobacterial repetitive intergenic consensus PCR. Siderophore production was measured by a colorimetric method. Biofilm formation was evaluated by a crystal violet assay. The results of the study showed that the expression of MDR is not significantly associated with an increase in these virulence factors or with biofilm formation. However, the origin of isolates had a significant association with siderophore gene availability and consequently on the concentrations of siderophores released. Genetic fingerprinting indicated that human and plant isolates have the same clonal origin, suggesting their circulation among humans and plants. CONCLUSION Antibiotic-susceptible strains of E. coli may be as virulent as MDR strains. Results also suggest that the environment can play a potential role in selection of strains with specific virulence factors. SIGNIFICANCE AND IMPACT OF THE STUDY Antibiotic-susceptible isolates of Escherichia coli from plant or human origin can be as virulent as the multidrug resistance (MDR) ones. Genetic relatedness was detected among the isolates of plant and human origin, indicating the circulation of these bacteria among human and plants. This could imply a potential role for environmental antimicrobial resistant bacteria in human infection.

Background Endogenous endophthalmitis caused by hypervirulent Klebsiella pneumoniae (HVKP) is an emerging infectious disease commonly with a devastating visual outcome. Most HVKP strains display a wild-type susceptibility profile to antibiotics. However, reports of antimicrobial-resistant HVKP have increased over time, which poses a serious therapeutic dilemma. Case presentation A 25-year-old man with a liver abscess and poorly controlled diabetes mellitus was admitted for endophthalmitis due to K. pneumoniae. The isolate displayed hypermucoviscosity as determined by a positive string test and exhibited resistance to ceftazidime, piperacillin-tazobactam and amikacin. Whole genome sequencing (WGS) analysis demonstrated the isolate to be a K1-serotype strain and belong to a novel single locus variant of ST23, ST2922. In addition to the virulence genes linked to HVKP, rmpA, magA, iucABCDiutA (aerobactin), ybtAPSTUX (yersiniabactin) and iroBDN (salmochelin), it was found to harbor extended-spectrum β-lactamase (ESBL) gene (bla CTX-M-14), AmpC β-lactamase gene (bla DHA), and 16S rRNA methylase gene (armA). Conclusions This is the first known case of endogenous endophthalmitis caused by a multidrug-resistant HVKP strain ever reported in China. Early diagnosis and treatment with intravenous and intravitreal injection of carbapenem were essential for a favorable visual outcome.

Iron is a vital mineral nutrient required by virtually all life forms to prosper; pathogenic bacteria are no exception. Despite the abundance of iron within the human host, highly regulated iron physiology can result in exceedingly low levels of iron bioavailable to prospective invading bacteria. To combat this scarcity of iron, many pathogenic bacteria have acquired specific and efficient iron acquisition systems, which allow them to thrive in iron-deficient host environments. One of the more prominent bacterial iron acquisition systems involves the synthesis, secretion, and reuptake of small-molecule iron chelators known as siderophores. Aerobactin, a citrate-hydroxamate siderophore originally isolated nearly 50 years ago, is produced by a number of pathogenic Gram-negative bacteria. Aerobactin has recently been demonstrated to play a pivotal role in mediating the enhanced virulence of a particularly invasive pathotype of Klebsiella pneumoniae (hvKP). Toward further understanding of this key virulence factor, we report the structural and functional characterization of aerobactin synthetase IucA from a strain of hvKP. The X-ray crystal structures of unliganded and ATP-bound forms of IucA were solved, forming the foundation of our structural analysis. Small angle X-ray scattering (SAXS) data suggest that, unlike its closest structurally characterized homologues, IucA adopts a tetrameric assembly in solution. Finally, we employed activity assays to investigate the substrate specificity and determine the apparent steady-state kinetic parameters of IucA.

Purpose: Escherichia coli isolated from asymptomatic bacteriuria (ABU) correlated genotypically and phenotypically with cystitis isolates may help in distinguishing urovirulence determinants from 'fitness factors', latter necessary only for survival of E. coli in urinary tract; for gaining insight into the pathogenesis of urinary tract infection. Materials and Methods: In this cross-sectional study, we compared genotypic (phylogroups and 15 putative virulence genes), and phenotypic profiles of ABU E. coli strains with our previously genotyped collection of cystitis isolates. Virulence score was calculated for each isolate as a number of virulence genes detected. Results: Significant differences were observed in the proportion of four phylogenetic groups (P = 0.009) amongst cystitis and ABU isolates. Average virulence score was higher for ABU isolates (6.6) than cystitis strains (4.2); and hlyA (P = 0.001), cytotoxic necrotising factor 1 (P = 0.00), fyuA (P = 0.00), ibeA (P = 0.00), kpsMII (P = 0.01), and malX/pathogenicity-associated island (P = 0.01) were more frequently present in ABU strains. Conclusions: The expression of adhesins, haemolysin, aerobactin, and capsule synthesis gene were similar in two groups suggesting their role as fitness factors. ABU isolates were better biofilm producers, reflecting its importance in silent persistence. Serum resistance gene which was more expressed in cystitis isolates may represent virulence determinant. Genetic makeup of E. coli does not change much rather genes helping in survival and colonisation are expressed equally in ABU and cystitis isolates as opposed to phenotypic attenuation of those that helps in invasion or inflammation in ABU isolates.

Uropathogenic Escherichia coli is the common pathogen to cause urinary tract infections (UTIs) and have become multidrug-resistant (MDR) extended-spectrum β-lactamase (ESBL) producers. The differences in the antimicrobial susceptibility, 5 bla genes, 12 virulence genes of 87 clinical ESBL-producing E. coli isolates and genomic variations and sequence types of 18 recurrent and repeated isolates from 9 patients were investigated. The 87 MDR-ESBL isolates collected mainly from indwelling urinary catheters (IUCs) and UTIs were highly resistant to fluoroquinolones, with over 50% of the isolates being resistant to cefepime and piperacillin/tazobactam and a few being resistant to carbapenem. These isolates carried at least two of the five bla genes examined, with the highest prevalence (87.4%) found for blaCTX-M (blaCTX-M3-like and blaCTX-M14-like), followed by blaCMY-2 (80.5%) and blaSHV (56.3%). The predominant virulence genes were the fimbriae gene fimH and the toxin genes cnf1 and hlyA in blood isolates and the capsule gene kpsMTII in UTI and blood isolates. Over 80% of the isolates carried yersiniabactin and aerobactin of siderophores. In 18 isolates, the fluoroquinolone-resistant ST131 isolate of pulsotypes I and II with blaCTX-M-15 was clonally disseminated in the hospital. The genomic plasticity of these ST131 occurred mainly through the conjugative plasmids with differences in replicon types A/C, I1, FIA, FIB and Y, size and number. In conclusion, MDR ESBL-producing E. coli isolates differed in virulence genes of UPEC and antibiotic resistance associated with the sources. Plasmid acquisition and chromosomal variations increase the spread of fluoroquinolone-resistant UPEC ST131 worldwide.

The relevance ofEscherichia coliassociated bacteriuria infection in pregnant women is poorly understood, despite these strains sharing a similar virulence profile with other extra intestinal pathogenicE. coliproducing severe obstetric and neonatal infections. We characterized and determined the antimicrobial susceptibility, resistant genes and virulence profiles of 82E. coliisolates associated with asymptomatic bacteriuria in some pregnant in five very distinct hospitals in the Volta region from January, 2016 to April, 2016 using Kirby-Bauer disc diffusion and polymerase chain reaction.High levels of antimicrobial resistance was observed to Ampicillin (79.3%), Tetracycline (70.7%) and Cotrimoxazole (59.8%), except for Cefuroxime (32.9%). Resistant genes analyses revealed 58.5% were positive forBlaTEMand 14.6% foraph(3)-Ia(aphA2). Virulence factors (VFs) was more widespread in pregnant women in the 2ndand 3rdtrimesters than 1sttrimester. VFs relating to adhesion (papC andiha), Protectins (traT), aerobactin acquisition (iutA) and iron acquisition systems (fyuA andirp2) were more prevalent in the resistantE. coliisolates. This study provides additional evidence for a link in bacteriuria and transmission of extra-intestinalE. coliin pregnant women to cause multi-resistant severe obstetric or neonatal infections. Considering the involvement of extra-intestinalE. coliin infections, our results may be helpful to develop strategies to prevent maternal and/ neonatal infections. In addition continuous surveillance is required to guide appropriate antibiotic usage in pregnant women.

Background A colistin-resistant mucoid Klebsiella strain was recovered from the blood of a patient in China. Hypervirulence has been reported in Klebsiella pneumoniae, but not in other Klebsiella spp. The strain was suspected to be hypervirulent and was therefore characterized. Methods The strain was subjected to genome sequencing using both the short-read Illumina HiSeq X10 Sequencer and the long-read MinION sequencer. Precise species identification was established using average nucleotide identity based on genome sequences. Virulence and antimicrobial resistance genes were identified using ResFinder and the bigsdb database. Conjugation experiments were performed. Virulence was assessed using wax moth (Galleria mellonella) larvae with control Klebsiella strains of low virulence and hypervirulence. Results The strain had a 5 553 341 bp circular chromosome and a 236 355 bp large plasmid. It was identified as Klebsiella variicola. The strain had multiple virulence genes encoding mucoid phenotype regulator (rmpA and rmpA2), aerobactin (iucABCD-iutA), salmochelin (iroBCDN) and yersiniabactin (irp1-2 and ybtAEPQSTUX) on the plasmid, which was not self-transmissible. It exhibited enhanced virulence in the larvae model, suggesting that the strain was hypervirulent. It was resistant to colistin (MIC = 8 mg/L) but was susceptible to amikacin, aztreonam, cefotaxime, ceftazidime, gentamicin, imipenem, meropenem, moxifloxacin, piperacillin/tazobactam, trimethoprim/sulfamethoxazole and tigecycline. The D150G substitution in PhoP, part of the PhoP-Q two-component system, which is known to mediate colistin resistance, was present in the strain. Conclusions Hypervirulence is not restricted to K. pneumoniae; it is also seen in other Klebsiella spp. The convergence of colistin resistance and hypervirulence in K. variicola represents a new challenge for health.

The increasing prevalence of KPC-producing Klebsiella pneumoniae strains in clinical settings has been largely attributed to dissemination of organisms of specific multilocus sequence types, such as ST258 and ST11. Compared with the ST258 clone, which is prevalent in North America and Europe, ST11 is common in China but information regarding its genetic features remains scarce. In this study, we performed detailed genetic characterization of ST11 K. pneumoniae strains by analyzing whole-genome sequences of 58 clinical strains collected from diverse geographic locations in China. The ST11 genomes were found to be highly heterogeneous and clustered into at least three major lineages based on the patterns of single-nucleotide polymorphisms. Exhibiting five different capsular types, these ST11 strains were found to harbor multiple resistance and virulence determinants such as the blaKPC-2 gene, which encodes carbapenemase, and the yersiniabactin-associated virulence genes irp, ybt and fyu. Moreover, genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA) were detectable in six genomes, whereas genes encoding salmochelin were found in three genomes. In conclusion, our data indicated that carriage of a wide range of resistance and virulence genes constitutes the underlying basis of the high level of prevalence of ST11 in clinical settings. Such findings provide insight into the development of novel strategies for prevention, diagnosis and treatment of K. pneumoniae infections.

Background Hypervirulent K. pneumoniae (hvKp) causes severe community acquired infections, predominantly in Asia. Though initially isolated from liver abscesses, they are now prevalent among invasive infections such as bacteraemia. There have been no studies reported till date on the prevalence and characterisation of hvKp in India. The objective of this study is to characterise the hypervirulent strains isolated from bacteraemic patients for determination of various virulence genes and resistance genes and also to investigate the difference between healthcare associated and community acquired hvKp with respect to clinical profile, antibiogram, clinical outcome and molecular epidemiology. Results Seven isolates that were susceptible to all of the first and second line antimicrobials and phenotypically identified by positive string test were included in the study. They were then confirmed genotypically by presence of rmpA and rmpA2 by PCR. Among the study isolates, four were from patients with healthcare associated infections; none were fatal. All patients with community acquired infection possessed chronic liver disease with fatal outcome. Genes encoding for siderophores such as aerobactin, enterobactin, yersiniabactin, allantoin metabolism and iron uptake were identified by whole genome sequencing. Five isolates belonged to K1 capsular type including one K. quasipneumoniae. None belonged to K2 capsular type. Four isolates belonged to the international clone ST23 among which three were health-care associated and possessed increased virulence genes. Two novel sequence types were identified in the study; K. pneumoniae belonging to ST2319 and K. quasipneumoniae belonging to ST2320. Seventh isolate belonged to ST420. Conclusion This is the first report on whole genome analysis of hypervirulent K. pneumoniae from India. The novel sequence types described in this study indicate that these strains are evolving and hvKp is now spread across various clonal types. Studies to monitor the prevalence of hvKp is needed since there is a potential for the community acquired isolates to develop multidrug resistance in hospital environment and may pose a major challenge for clinical management.

Klebsiella pneumoniae strains that are commonly recognized by clinicians and microbiologists are termed as classical K. pneumoniae (cKP). A strain with capsule-associated mucopolysaccharide web is known as hypervirulent K. pneumoniae (hvKP) as it enhances the serum resistant and biofilm production. Aim is to determine and correlate various virulence genes, ESBL, serum bactericidal effect and biofilm formation of clinical isolated cKP and hvKP from respiratory tract infected patients. A total of 96 K. pneumoniae strains were isolated from sputum of respiratory tract infected patients. The isolates were performed string test, AST, ESBL virulence gene, serum bactericidal and biofilm assays. Out of 96 isolates, 39 isolates (40.6%) were identified with hypervirulent phenotypes. The number of cKP exhibiting resistance to the tested antimicrobials and ESBLs were significantly higher than that of the hvKP strains. The virulence genes of K. pneumoniae such as K1, K2, rmpA, uge, kfu and aerobactin were strongly associated with hvKP than cKP. However, no significant difference was found in FIM-1 and MrKD3 genes. ESBL producing cKP and hvKP were significantly associated with strong biofilm formation (both P < 0.05) and highly associated with bactericidal effect of serum (both P < 0.05) than cKP strains. However, neither biofilm formation nor bactericidal effect of serum was found with significant difference in between ESBL producing cKP and ESBL producing hvKP strains (both P > 0.05). Although the hvKP possess more virulence gene, but they didn’t show any significant difference between biofilm formation and bactericidal effect of serum compared with ESBL producing cKP strains.

The expression of aerobactin accounts for much of the pathogenesis of avian pathogenic Escherichia coli (APEC). iucA, iucB, iucC and iucD are involved in aerobactin synthesis and iutA is responsible for the expression of a specific outer membrane receptor protein for ferric aerobactin. Knockout mutants of iucD and iucDiutA in the APEC O2 strain E058 were constructed and named E058ΔiucD and E058ΔiucDΔiutA, respectively. To evaluate the pathogenicity of these mutants, we used multiple approaches to assess the effects of mutations on the virulence of APEC E058. Aerobactin-defective mutants E058ΔiucD and E058ΔiucDΔiutA showed significantly decreased pathogenicity compared with the wild-type strain E058, evidenced by the low extent of colonization in selected organs or being outcompeted by E058 in vivo. Chickens challenged with APEC E058 exhibited typical signs and lesions of avian colibacillosis, while those inoculated with the mutants E058ΔiucD or E058ΔiucDΔiutA showed moderate airsacculitis, mild pericarditis and perihepatitis. However, no significant differences in resistance to specific-pathogen-free chicken serum, ingestion by HD-11 cells, and growth rates in LB were observed between the mutants and the wild-type strain. These results indicated that the IucD- and IutA-related virulence factors play a significant role in the pathogenesis of the APEC strain E058.

Foodstuffs of animal origin may present hazards, due to bacterial contamination. This study was conducted to determine Escherichia coli in some types of animal source foods (raw milk, raw beef meat and raw poultry meat). Bacteriological examination of total 246 raw food samples of animal origin, raw milk, (105); beef meat (31) and poultry meat (110) were collected from different localities in Ismailia City showed that 64/246 (26.01%) of samples were infected with E. coli. Serological identification of (10) E. colistrains revealed that they were belonged to O111 polyvalent 1; O44 polyvalent 2 (2 strains for each); O78 polyvalent 4; O146 polyvalent 2; O26 polyvalent 1; O119 polyvalent 1; O86 polyvalent 1 and O18 polyvalent 3 (1 strain for each). E. coli strains showed high susceptible rate to Ciprofloxacin (CIP), (100%) and high resistance (100%) to Ampicillin (AMP), Metronidazole (MTZ), Amoxicillin (AML), and Vancomycin (VAS). Four strains of the isolated E. coli were submitted to molecular studies for detection of the eaeA (protein intimin), iutA (encoding aerobactin) and iss (increased serum survival protein) genes, by using PCR technique.