Bla DHA-1 Research Articles

Emergence and genomic characteristics of multi-drug-resistant Salmonella in pet turtles and children with diarrhoea.

Pet turtles are a well-recognized source of human salmonellosis, posing a threat to human health, particularly children who commonly keep pet turtles. To date, the genomic characteristics of Salmonella among pet turtles and children has not been well described. We investigated the prevalence, antimicrobial resistance (AMR) and genomic characteristics of Salmonella from pet turtles in Beijing, China. In total, 9.6 % (46/480) of pet turtles were positive for Salmonella with S. Thompson being the dominant serovar (19/46) in 2019. Moreover, 80.4 % of Salmonella were multi-drug resistant (MDR) and 60.7 % were resistant to ampicillin, streptomycin, sulfonamides and tetracycline (ASSuT). We further compared the genomes of S. Thompson isolates from pet turtles (n=19) with those from children with diarrhoea (n=28) in the same region and year, most of which were sequence type (ST)26, with one novel ST7937 identified from a child-associated isolate. S. Thompson isolates from children with diarrhoea exhibited less resistance than isolates from pet turtles. Most MDR isolates possessed multiple AMR genes, including the AmpC β-lactamase-encoding genes bla DHA-15 and bla DHA-1 which co-occurred with the IncA/C and IncHI plasmid replicon types. To the best of our knowledge, this is the first time that the bla DHA-15 gene has been detected from Salmonella. Several pet turtle-associated S. Thompson isolates comprised phylogenetically close clusters with those from children with diarrhoea (<20 SNP differences). Bayesian analysis demonstrated that the Chinese ST26 S. Thompson strains had a recent evolutionary history and evolved into two major clades, with one clade acquiring various resistant plasmids. Our findings revealed the emergence of MDR Salmonella among pet turtles in China and provided evidence for the interspecies transmission of S. Thompson.

Genomic characterisation of Escherichia coli isolated from poultry at retail through Sink Surveillance in Dhaka, Bangladesh reveals high levels of multi-drug resistance.

The surveillance of antimicrobial resistance (AMR) in commensal Escherichia coli from livestock at slaughter is widely employed to assess the potential for risk to humans. There is currently a limited understanding of AMR in Bangladesh poultry at retail in live bird markets, with studies focussing solely on phenotypic characterisation of resistance. To address this evidence gap we performed antimicrobial susceptibility testing and whole genome sequencing on E. coli obtained from chickens from live bird markets in Dhaka in 2018 (n = 38) and 2020 (n = 45). E. coli were isolated from caeca samples following ISO guidelines and sequenced using short and long read methods. Multidrug resistance was extremely common (n = 77) and there was excellent concordance between AMR phenotype and the presence of corresponding AMR genes or mutations. There was considerable genomic diversity, with 43 different sequence types detected. Public health considerations included the high occurrence of resistance to ciprofloxacin (n = 75) associated with plasmid-residing qnrS or mutations in the gyrA and parC chromosomal genes; and the detection of a tigecycline resistant isolate harbouring tet(X4) on an IncHI1A/B-IncFIA mosaic plasmid. Thirty-nine isolates were resistant to azithromycin and harboured mphA, with a significant increase in the incidence of resistance between 2018 and 2020. Although azithromycin is banned for veterinary use in Bangladesh it remains an important treatment option for humans. Interestingly, mphA confers high-level resistance to azithromycin and erythromycin, and the latter is commonly used on poultry farms in Bangladesh. Seven isolates were colistin resistant and carried mcr1. For two isolates hybrid assemblies revealed that mcr1 resided on a highly conserved IncHI2 plasmid that had 93% nucleotide identity to a plasmid from the published genome of an E. coli isolate of Bangladeshi human origin. Six isolates had resistance to third generation cephalosporins, associated with plasmid-residing bla CTX-M-55, bla CTX-M-65, or bla DHA-1. By employing phenotypic and genomic approaches for AMR surveillance we have provided new insights into the potential for One Health AMR linkages in Bangladesh. Employing similar approaches in human and environmental sectors will help inform the One Health approach to addressing AMR, and generate evidence to support mitigation measures such as improved antimicrobial stewardship.

Occurrence and characterization of plasmids carrying tmexCD1-toprJ1, blaDHA-1, and blaCTX-M-127, in clinical Klebsiella pneumoniae strains.

Today, the emergence of Klebsiella pneumoniae with the tmexCD1-toprJ1 gene cassette in patients has presented a significant clinical challenge. To present the detailed genetic features of the tmexCD1-toprJ1 gene cassette of K. pneumoniae strain F4_plasmid pA, the whole bacterial genome was sequenced by Illumina and nanopore platforms, and mobile genetic elements related to antibiotic resistance genes were analyzed with a series of bioinformatics methods. K. pneumoniae strain F4 was determined to be a class A+C beta-lactamase, and was resistant to routinely used antibiotics, especially tigecycline, because of the oqxAB gene localized on the F4_chromosome and tmexCD1-toprJ1 on F4_plasmid A. After plasmid transfer assays, the F4_plasmid pA or F4_plasmid pB could be recovered with an average conjugation frequencies of 3.42×10-4 or 4.19×10-4. F4_plasmid pA carried tmexCD1-toprJ1 and bla DHA-1 accompanied by genetic intermixing of TnAs1, Tn5393, TnAs3, and In641, while F4_plasmid pB, bearing bla CTX-M-174, had structural overlap of TnAs3 and In641. We suggested that plasmids carrying tmexCD1- toprJ1 might be strongly related to IS26-integrated loop intermediates. This study showed that due to the structural evolution of F4 and related strains, their resistances were so strong that effective antibiotics were virtually unavailable, therefore their spread and prevalence should be strictly controlled.

Characterization of beta-lactamase producing Enterobacterales isolated from an urban community wastewater treatment plant in Iran.

he occurrence and characteristics of Extended Spectrum- and AmpC-β-lactamase producing Enterobacterales (ESBL-PE and AmpC-PE) in an urban wastewater treatment plant (WWTP) were investigated. A total of 30 wastewater samples were collected from all sections of WWTP. Enterobacterales were isolated and identified using standard microbiological tests. The antibiotic resistance profile was determined by the Kirby-Bauer disk diffusion method. Phenotypic screening for ESBL-PE and AmpC-PE isolates was performed by double-disk synergy and boronic acid disk potentiation tests, respectively. The isolates were examined for AmpC- and ESBL-encoding genes by PCR and sequencing methods. Among 146 Enterobacterales isolates, 8.9% (n=13) [ESBL-only; 5.48% (n=8) and ESBL + AmpC; 3.42% (n=5)] were ESBL-producers and 15.75% (n=23) [AmpC-only; 12.33% (n=18) and ESBL + AmpC; 3.42% (n=5)] AmpC-producers. Hafnia spp. with 33.33% (n=1/3) and E. coli with 20.58% (n=7/34) [ESBL-only; 17.64% (n=6/34) and ESBL + AmpC; 2.94% (n=1/34)] were the most common ESBL-producing bacteria. Enterobacter spp. with 37.50% (n=6/16) of isolates were the most common AmpC-producing organisms. ESBL- and/or AmpC-producing isolates were identified in all parts of the WWTP including 80% (n=8/10) of samples taken from effluent. Among ESBL-producing isolates, bla CTX-M , bla TEM, and bla SHV ESBL-encoding genes were found in 61.5% (n=8), 15.3% (n=2), and 7.7% (n=1) of isolates, respectively. All CTX-M-type enzymes belonged to the CTX-M-1 group and CTX-M-15 subgroup. bla TEM and bla SHV type genes belonged to bla TEM-20 and bla HSV-12 subtypes, respectively. bla DHA with 73.9% (n=17/23), and bla CIT and bla FOX with 30.4% (n=7/23) each, were the most common AmpC-encoding genes among AmpC-producing isolates. Overall, 75% of ESBL-producing and 55.5% of AmpC-producing isolates exhibited multi-drug resistance phenotypes. The organisms were most resistant against ampicillin (82.2%) nalidixic acid (43.8%) and cephalexin (41.1%). ESBL- and AmpC-producing Enterobacterales spp. with diverse genetic resistance backgrounds in WWTP effluent poses a significant risk to public health.

Genomic Characterization of ESBL/AmpC-Producing Escherichia coli in Stray Dogs Sheltered in Yangzhou, China.

Limited data are available on the prevalence and antimicrobial resistance of extended spectrum β-lactamase- (ESBL) and AmpC β-lactamase-producing Escherichia coli in stray dogs. We aimed to investigate the genomic characteristics of ESBL/AmpC-producing E. coli isolated from stray dogs sheltered in Yangzhou, China. We collected 156 samples including 115 fecal swabs, 35 kennel floor swabs, two breeder hand and shoe sole swabs, and four feed samples. The isolates were tested for resistance by antimicrobial susceptibility testing and further analyzed for cefotaxime-resistant E. coli isolates by whole genome sequencing. We identified 80 cefotaxime-resistant E. coli isolates (51.3%), 59 isolates (73.8%) from feces and 21 (26.2%) from the environment. Whole-genome sequencing analysis showed that bla CTX-M-15 (n=30) and bla CTX-M-55 (n=29) were the most prevalent genotypes. Two isolates only carried the AmpC β-lactamase gene bla CMY-2; one isolate had a combination of AmpC β-lactamase gene bla DHA-1 and ESBL β-lactamase gene bla CTX-M-14. Other important resistance genes such as bla OXA-10, bla TEM-1B, bla TEM-135, bla TEM-106, tet(A), qnrS1, qnrB4, and oqxAB were also detected. The serotype combination was highly abundant, with O10:H25 predominating (n=12). Most cefotaxime-resistant E. coli isolates belonged to phylogroup A (62.5%, n=50), followed by phylogroup B1 (26.3%, n=21). Thirty different sequence types (STs) and 27 distinct plasmid replicons were identified, among which ST2325 (n=12) and IncFII (n=38) was the most frequent ST and plasmid, respectively. ESBL/AmpC-producing isolates were divided into four major clades; clade IV was the primary lineage containing 37 isolates from feces and 13 from the environment. Three high-risk E. coli clone ST23 strains and one ST10 strain belonged to clades III and IV, respectively. Our study provides a comprehensive overview of resistance profiles and genomic characteristics in ESBL/AmpC-producing E. coli and highlights the possible role of stray dogs as an antibiotic resistance gene reservoir.

Clinical and resistance characterization of carbapenem-resistant Klebsiella pneumoniae isolated from intensive care units in China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious threat to health, and the detection rate in intensive care units (ICUs) is relatively high. We compared regional differences in the clinical and molecular characteristics of CRKP from three ICUs in different hospitals, to make a reference and contribution for infection control and clinical medication. A total of 150 CRKP strains from Chongqing, Beijing, and Nantong, as well as the clinical data of the infected patients, were collected between 2019 and 2021. The carbapenemase phenotype was determined by CarbaNP test, and the outer membrane porin (OMP) genes (ompK35/ompK36), multi-locus sequence typing (MLST) and resistance genes were identified by polymerase chain reaction (PCR) amplification and sequencing. Patients infected with CRKP were mainly elderly, with comorbidity, and had undergone invasive operation and multiple antibiotic therapy. All strains exhibited high-level resistance to most antibiotics except for polymyxin B and tigecycline. Among the CRKP strains, 100 had the bla KPC-2 gene and 8 had bla NDM-1 gene, which were distributed in all of the hospitals. Nearly all the strains harbored extended-spectrum beta-lactamase (ESBL) genes (bla SHV, bla CTX-M, and bla TEM). Class C carbapenemase genes (bla CIT, bla DHA), and deletion and mutation of ompK35/ompK36 existed in some strains. ST11 was the main MLST type, followed by ST15. There were a few significant differences in the molecular epidemiology and clinical characteristics, but generally the features of CRKP from the three ICUs aligned fairly well, which might have resulted from dissemination through frequent personnel exchanges between regions.

Fecal Carriage of Extended-Spectrum β-Lactamases and pAmpC Producing Enterobacterales in an Iranian Community: Prevalence, Risk Factors, Molecular Epidemiology, and Antibiotic Resistance.

This study aimed to determine the prevalence and risk factors associated with intestinal carriage of extended-spectrum β-lactamases producing Enterobacterales (ESBL-PE) and plasmid-mediated AmpC β-lactamase producing Enterobacterales (AmpC-PE) in healthy children in Ardabil, Iran. A total of 305 fecal samples were collected. Isolates underwent antimicrobial susceptibility testing, phenotypic and genotypic identification of β-lactamase production, and epidemiologic molecular typing. In total, 21.5%, 1.5%, and 1.2% of volunteers were extended-spectrum β-lactamase (ESBL)-, AmpC-, and simultaneous ESBL/AmpC-PE carriers, respectively. Escherichia coli was the predominant ESBL producing bacterium (70.2%) found in ESBL-PE colonized subjects. Beyond ESBL positive isolates, bla CTX-M group genes were the most common type (75.6%) and bla TEM (non-bla TEM-1 and non- bla TEM-2) were in the second place (25.6%). Among bla CTX-M genes, bla CTX-M-1 (55.3%) and bla CTX-M-15 (55.3%) were the most predominant types with equal prevalence. Some isolates were multi-enzyme producers. bla CIT and bla DHA genes were common AmpC type enzyme encoding genes found in AmpC-PE isolates. Most isolates produced both enzymes at the same time. The number of students in the classes was statistically associated with ESBL-PE intestinal carriage (p < 0.05). Moreover, 46 (65.7%), 3 (60%), 4 (100%), and 98 (39.8%) ESBL-, AmpC-, ESBL/AmpC, and non-ESBL/AmpC-PE isolates were multidrug-resistant, respectively. Overall, regardless of β-lactam antibiotics, 62% and 59.5% of isolates were resistant to co-trimoxazole and tetracycline, respectively. The majority of ESBL producing E. coli isolates (69.2%) belonged to phylogroup A. According to Enterobacterial repetitive intergenic consensus polymerase chain reaction, there was no clonal relatedness between isolates. This study showed a high rate of multi-resistant ESBL-PE intestinal carriage among healthy individuals in Iran.

Nasal carriage of CTX-M-55-producing Escherichia coli ST8369 in a healthy cohort in the city of Yangzhou, China.

This study aimed to investigate the prevalence and diversity of extended-spectrum β-lactamases (ESBL)-producing Escherichia coli isolates from healthy individuals in a community and to elucidate their dissemination mechanism. Cefotaxime-resistant E. coli were isolated from 95 samples of healthy persons from one community in Yangzhou, China, and were tested for minimal inhibitory concentrations of 14 antimicrobial agents. The isolates were subjected to whole genome sequencing by Illumina Hiseq or PacBio single-molecule real-time sequencing. A total of 30 cefotaxime-resistant E. coli isolates were obtained, carrying bla CTX-M (n=29) or bla DHA (n=1), of which the bla CTX-M-55 (n=19) was the most predominant genotype. One novel bla CTX-M variant bla CTX-M-252 was identified. Thirteen CTX-M-55-producing E. coli isolates belonged to ST8369 from nasal (n=12) or faecal (n=1) samples shared the identical cgMLST type, resistance profiles, resistance genes, plasmid replicons, and a 5,053-bp bla CTX-M-55 structure ΔIS26-ΔISEcp1-bla CTX-M-55-Δorf477-ΔTn2. The bla CTX-M-55 gene was located on IncHI2/ST3 plasmid in E. coli ST8369. The lengths of bla CTX-M/bla DHA-carrying contigs in the remaining 17 E. coli strains ranged from 1,663 to 382,836 bp, located on chromosome (n=4) or plasmids (n=5); the location of the other eight contigs could not be determined due to incomplete assembly. The bla CTX-M was associated with ISEcp1 as previously reported. Nasal colonization of CTX-M-55-producing ST8369 E. coli strains has occurred among healthy individuals in one community. There is a potential risk of antimicrobial resistance dissemination between humans within one community through close contact or environment via aerosols or dust. Therefore, surveillance of nasal carriage of bla CTX-M in communities is warranted to further monitor the spread of the antimicrobial resistance genes in China.

Antimicrobial susceptibility, plasmid replicon typing, phylogenetic grouping, and virulence potential of avian pathogenic and faecal Escherichia coli isolated from meat chickens in Australia

ABSTRACT Globally, avian colibacillosis is a leading cause of morbidity and mortality in poultry, associated with economic losses and welfare problems. Here, clinical avian pathogenic E. coli isolates (CEC; n = 50) and faecal E. coli isolates from healthy (FEC; n = 187) Australian meat chickens collected between 2006 and 2014 were subjected to antimicrobial susceptibility testing, phylogenetic grouping, plasmid replicon (PR) typing, multilocus sequence typing, and virulence gene (VG) profiling. Extended-spectrum cephalosporin (ESC)- and fluoroquinolone (FQ)-resistant E. coli isolates underwent further genetic characterization. Significant proportions of CEC and FEC were, respectively, susceptible (13/50; 48/187) or MDR (9/50; 26/187) to 20 tested antimicrobials. Phylogenetic groups A and C, and PR types IncFIB and IncFrep were most represented. Five tested CEC-associated VGs were more prevalent in CEC (≥ 90%) than FEC (≤ 58%). Some isolates (CEC n = 3; FEC n = 7) were resistant to ESCs and/or FQs and possessed signature mutations in chromosomal FQ target genes and plasmid-mediated qnrS, bla CMY-2, and bla DHA-1 genes. Sequence type 354 (n = 4), associated with extraintestinal infections in a broad range of hosts, was prevalent among ESC- and/or FQ-resistant FEC. This study confirmed existence of a small reservoir of ESC- and FQ-resistant E. coli in Australian commercial meat chickens despite absence of use in the industry of these drugs. Otherwise, diversity of VGs and PR types in both FEC and CEC populations was identified. We hypothesize that the source of ESC- and FQ-resistant E. coli is external to poultry production facilities. RESEARCH HIGHLIGHTS Low-level resistance to older and newer generation antimicrobial drugs detected. The most common sequence type (ST) associated with FQ resistance was ST354 (4/10). A small proportion of CEC (n = 3) and FEC (n = 7) were resistant to ESCs and/or FQs.

β-lactamases (bla TEM, bla SHV, bla CTXM-1, bla VEB, bla OXA-1 ) and class C β-lactamases gene frequency in Pseudomonas aeruginosa isolated from various clinical specimens in Khartoum State, Sudan: a cross sectional study.

Background:Pseudomonas aeruginosa is a pathogenic bacterium, causing nosocomial infections with intrinsic and acquired resistance mechanisms to a large group of antibiotics, including β-lactams. This study aimed to determine the susceptibility pattern to selected antibiotics and to index the first reported β-lactamases genes frequency in Ps. aeruginosa in Khartoum State, Sudan. Methods: 121 Ps. aeruginosa clinical isolates from various clinical specimens were used in this cross sectional study conducted in Khartoum State. Eighty isolates were confirmed as Ps.aeruginosa through conventional identification methods and species specific primers. The susceptibility pattern of the confirmed isolates to selected antibiotics was done following the Kirby Bauer disk diffusion method. Multiplex PCR was used for detection of seven β-lactamase genes ( blaTEM, blaSHV, blaCTXM-1, blaVEB, blaOXA-1, blaAmpC and blaDHA). Results: Of the 80 confirmed Ps. aeruginosa isolates, 8 (10%) were resistant to Imipenem while all isolates were resistant to Amoxicillin and Amoxyclav (100%). A total of 43 (54%) Ps. aeruginosa isolates were positive for blaTEM, blaSHV, blaCTXM-1, blaVEB and blaOXA-1 genes, while 27 (34%) were positive for class C β- Lactamases, and 20 (25%) were positive for both classes. Frequency of beta-lactamases genes was as follows: blaTEM, 19 (44.2%); blaSHV, 16 (37.2%); bla CTX-M1, 10 (23.3%); blaVEB, 14 (32.6%); blaOXA-1, 7 (16.3%). blaAmpC 22 (81.5%) and bla DHA 8 (29.6%). In total, 3 (11.1%) isolates were positive for both bla AmpC and blaDHA genes. Conclusion:Ps. aeruginosa isolates showed a high rate of β- lactamases production, with co-resistance to other antibiotic classes. The lowest resistance rate of Ps. aeruginosa was to Imipenem followed by Gentamicin and Ciprofloxacin. No statistically significant relationship between production of β-lactamases in Ps. aeruginosa and resistance to third generation cephalosporins was found.

Hypervirulent and hypermucoviscous extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Klebsiella variicola in Chile

ABSTRACT Convergence of virulence and antibiotic-resistance has been reported in Klebsiella pneumoniae, but not in Klebsiella variicola. We, hereby, report the detection and genomic characterization of hypervirulent and hypermucoviscous K. pneumoniae and K. variicola recovered in Chile from health-care associated infections, which displayed resistance to broad-spectrum cephalosporins. One hundred forty-six K. pneumoniae complex isolates were screened by hypermucoviscosity by the “string test.” Two hypermucoid isolates, one hypermucoviscous K. pneumoniae (hmKp) and one K. variicola (hmKv), were further investigated by whole-genome sequencing. In vivo virulence was analyzed by the Galleria mellonella killing assay. In silico analysis of hmKp UCO-494 and hmKv UCO-495 revealed the presence of multiple antibiotic-resistance genes, such as bla CTX-M-1, bla DHA-1 and bla LEN-25 among others clinically relevant resistance determinants, including mutations in a two-component regulatory system related to colistin resistance. These genetic features confer a multidrug-resistant (MDR) phenotype in both strains. Moreover, virulome in silico analysis confirmed the presence of the aerobactin gene iutA, in addition to yersiniabactin and/or colicin V encoding genes, which are normally associated to high virulence in humans. Furthermore, both isolates were able to kill G. mellonella and displayed higher virulence in comparison with the control strain. In summary, the convergence of virulence and the MDR-phenotype in K. pneumoniae complex members is reported for the first time in Chile, denoting a clinical problem that deserves special attention and continuous surveillance in South America.

Hybrid genome assembly of Shigella sonnei reveals the novel finding of chromosomal integration of an IncFII plasmid carrying a mphA gene.

Azithromycin is increasingly being used for the treatment of shigellosis despite a lack of interpretative guidelines and with limited clinical evidence. The present study determined azithromycin susceptibility and correlated this with macrolide-resistance genes in Shigella spp. isolated from stool specimens in Vellore, India. The susceptibility of 332 Shigella isolates to azithromycin was determined using the disc diffusion method. Of these, 31 isolates were found to be azithromycin resistant. The azithromycin minimum inhibitory concentration (MIC) was determined using the broth microdilution method. In addition, isolates were screened for mphA and ermB genes using conventional PCR. Furthermore, an isolate that was positive for resistance genes was subjected to complete genome analysis, and was analysed for mobile genetic elements. The azithromycin MIC for the 31 resistant Shigella isolates ranged between 2 and 16 mg l−1. PCR results showed that a single isolate of Shigella sonnei carried a mphA gene. Complete genome analysis revealed integration of an IncFII plasmid into the chromosome of S. sonnei , which was also found to carry the following resistance genes: sul1, bla DHA1, qnrB4, mphA, tetR. Mutations in the quinolone-resistance-determining region (QRDR) were also observed. Additionally, prophages, insertion sequences and integrons were identified. The novel finding of IncFII plasmid integration into the chromosome of S. sonnei highlights the potential risk of Shigella spp. becoming resistance to azithromycin in the future. These suggests that it is imperative to monitor Shigella susceptibility and to study the resistance mechanism of Shigella to azithromycin considering the limited treatment choices for shigellosis.

β-lactamases (bla TEM, bla SHV, bla CTXM-1, bla VEB, bla OXA-1) and class C β-lactamases gene frequency in Pseudomonas aeruginosa isolated from various clinical specimens in Khartoum State, Sudan: a cross sectional study

Background: Pseudomonas aeruginosa is a pathogenic bacterium, causing nosocomial infections with intrinsic and acquired resistance mechanisms to a large group of antibiotics, including β-lactams. This study aimed to determine the susceptibility pattern to selected antibiotics and to index the first reported β-lactamases genes frequency in Ps. aeruginosa in Khartoum State, Sudan. Methods: 121 Ps. aeruginosa clinical isolates from various clinical specimens were used in this cross sectional study conducted in Khartoum State. Eighty isolates were confirmed as Ps. aeruginosa through conventional identification methods and species specific primers. The susceptibility pattern of the confirmed isolates to selected antibiotics was done following the Kirby Bauer disk diffusion method. Multiplex PCR was used for detection of seven β-lactamase genes ( blaTEM, blaSHV, blaCTXM-1, blaVEB, blaOXA-1, blaAmpC and blaDHA). Results: Of the 80 confirmed Ps. aeruginosa isolates, 8 (10%) were resistant to Imipenem while all isolates were resistant to Amoxicillin and Amoxyclav (100%). A total of 43 (54%) Ps. aeruginosa isolates were positive for blaTEM, blaSHV, blaCTXM-1, blaVEB and blaOXA-1 genes, while 27 (34%) were positive for class C β- Lactamases, and 20 (25%) were positive for both classes. Frequency of beta-lactamases genes was as follows: blaTEM, 19 (44.2%); blaSHV, 16 (37.2%); bla CTX-M1, 10 (23.3%); blaVEB, 14 (32.6%); blaOXA-1, 7 (16.3%). blaAmpC 22 (81.5%) and bla DHA 8 (29.6%). In total, 3 (11.1%) isolates were positive for both bla AmpC and blaDHA genes. Conclusion: Ps. aeruginosa isolates showed a high rate of β- lactamases production, with co-resistance to other antibiotic classes. The lowest resistance rate of Ps. aeruginosa was to Imipenem followed by Gentamicin and Ciprofloxacin. No statistically significant relationship between production of β-lactamases in Ps. aeruginosa and resistance to third generation cephalosporins was found.

Multidrug-resistant Escherichia coli isolated from a dog with a history of urolithiasis: case report

ABSTRACT Bacterial resistance is a reality in both human and veterinary health, it limits the therapeutic arsenal and raises the costs of the patient’s treatment. A dog with signs of cystitis received treatment with 5mg/kg enrofloxacin at three consecutive times, with low effectiveness. The presence of urethral uroliths was identified and urohydropulsion was done. The animal presented a new obstruction, for which a cystotomy was performed, but continued with signs of infection. Uroculture and antimicrobial susceptibility test were then performed. Escherichia coli was identified, which was resistant to 13 antibiotics, being sensitive only to piperacillin-tazobactam and amikacin. In the screening test for β-lactamase, the production of ESβL was detected. The qPCR indicated the presence of the bla CTXm, bla DHA, bla OXA, bla IMP, bla TEM, bla GIM, bla SIM, bla SPM and bla SME genes, which may lead to a phenotypic resistance profile for ampicillin, amoxicillin-clavulanate, aztreonam, cefepime cefoxitin, cefuroxime, ceftazidime, ceftriaxone, imipenem, and piperacillin-tazobactam. This case reaffirms the value that laboratory analysis adds to the diagnosis and treatment of cystitis and urolithiasis, which can define the direction of evolution of the prognosis and the speed at which the patient's health will be restored.

Virulence characterization of Klebsiella pneumoniae and its relation with ESBL and AmpC beta-lactamase associated resistance

Trend analysis reveals that Klebsiella pneumoniae has witnessed a steep enhancement in the antibiotic resistance and virulence over the last few decades. The present investigation aimed at a comprehensive approach investigating antibiotic susceptibility including, extended spectrum beta-lactamase (ESBL) and AmpC β-lactamase (AmpC) resistance and the prevalence of virulence genes among the K. pneumoniae isolates. Sixty-one K. pneumoniae isolates were obtained from various clinical infections. Antimicrobial susceptibility was performed by disk diffusion method. The Mast® D68C test detected the presence of ESBLs and AmpCs phenotypically, and later presence of ESBL and AmpC genes was observed by polymerase chain reaction (PCR). Multiplex-PCR was performed to investigate various virulence genes. Amongst 61 K. pneumoniae isolates, 59% were observed as ESBL and 14.7% as AmpC producers. All ESBL producers were positive for bla CTX-M-15 , while bla CTX-M-14 was observed in 54.1% isolates. The frequency of AmpC genes was as follows: bla CMY-2 (60.7%) and bla DHA-1 (34.4%). The most frequent virulence genes were those encoding enterobactin and lipopolysaccharide. Presence of mrkD was associated with bla DHA-1 gene, while bla CMY-2 significantly (p≤0.05) correlated with the presence of iutA and rmpA virulence genes. bla DHA-1 positive isolates had urine as a significant source, while bla CMY-2 positive isolates were mainly collected from wound exudates (p≤0.05). Our results highlight that ESBL and AmpC production along with a plethora of virulence trait on K. pneumoniae should be adequately considered to assess its pathogenesis and antibiotic resistance.

Draft genome sequencing and annotation of a low-virulence Morganella morganii strain CQ-M7, a multidrug-resistant isolate from the giant salamander in China

A multidrug-resistant Morganella morganii strain (CQ-M7), isolated from the kidney of a diseased Chinese giant salamander in China, was examined with whole genome sequencing to better understand drug tolerance and its pathogenicity. The draft genome of the investigated strain was assembled using HGA assembler and annotated using Rapid Annotations Subsystems Technology (RAST) server. The contigs were annotated by the appropriate bioinformatics tools available on the National Center for Biotechnology Information (NCBI) website. Antibiotic resistance genes were detected by PCR. Pathogenicity of the isolate was performed on 30 healthy Chinese giant salamanders with different infection dosages. The CQ-M7 strain showed resistance to multiple antimicrobials, especially to aminoglycoside and β-lactam antibiotics. Seventeen drug-resistance genes were detected, which were related to β-lactams, aminoglycosides, fluoroquinolones, tetracyclines, peptide antibiotic, and fosfomycin resistance. Sequence analysis showed the assembled genome size to be 4 966 326bp with 51.16% of GC content, containing 4587 protein-coding genes, 71 pseudogenes, five rRNAs, 80 tRNAs, and five noncoding RNAs. The genome sequence was deposited in GenBank under accession number RQIJ00000000. Artificial infection results indicated that the CQ-M7 strain was a low-virulence strain for the Chinese giant salamander. It is believed that this is the first draft genome of Chinese giant salamander original Morganella morganii strain harbouring multiple antibiotic resistance genes in China. The reported genome sequence could provide insights into antibiotic resistance mechanisms and control strategies of Morganella morganii.

P1220-CTXM, a pKP048-related IncFIIK plasmid carrying bla CTX-M-14 and qnrB4.

This study aimed to characterize plasmid-mediated antimicrobial resistance in clinical Klebsiella pneumoniae 1220 carrying bla CTX-M-14 and qnrB4. Plasmid p1220-CTXM was transformed from the 1220 isolate into Escherichia coli through conjugal transfer and then fully sequenced. Antimicrobial susceptibility was determined by VITEK. p1220-CTXM was an IncFIIK plasmid genetically closely related to pKP048 and carried resistance markers including bla CTX-M-14, bla DHA-1, qnrB4, sul1 and qacEΔ1, all of which were harbored in a 35.7-kb multidrug-resistant region. bla CTX-M-14 was located in a truncated ISEcp1-bla CTX-M-14-orf477 transposition unit, and qnrB4 and bla DHA-1 were in a truncated qnrB4-bla DHA-1 region. This study provided the insight into the co-occurrence of bla CTX-M-14 and qnrB4 and the evolution of pKP048-related IncFIIK plasmids.

Countrywide dissemination of a DHA-1-type plasmid-mediated AmpC β-lactamase-producing Klebsiella pneumoniae ST11 international high-risk clone in Hungary, 2009–2013

The first plasmid-mediated AmpC β-lactamase-producing Klebsiella pneumoniae (pAmpC KP) isolate was detected in December 2009 in Hungary. Hungarian microbiological laboratories were asked to send all KP strains showing cefoxitin resistance and decreased susceptibility or resistance to any third-generation cephalosporins to the Reference Laboratories at the National Center for Epidemiology. Investigation was conducted in order to outline spatio-temporal distribution and genetic characterization of pAmpC-KP isolates in Hungary. Between December 2009 and December 2013, 312 consecutive KP clinical isolates were confirmed as producing pAmpCs. All isolates showed resistance to third-generation cephalosporins, aminoglycosides and fluoroquinolones, and 77 % were non-susceptible to at least one carbapenem. Analysis of β-lactamase genes showed blaDHA-1 in all and additionally blaCTX-M-15 in 90 % of isolates. PFGE typing revealed 12 pulsotypes; of these, KP053 (262/312) and KP070 (38/312) belonged to sequence type ST11 and comprised 96 % of the isolates. The blaDHA-1 and blaCTX-M-15 co-producing KP053/ST11 clone affected 234 patients and spread to 55 healthcare centres across Hungary during the study period. Three KP053 isolates were also resistant to colistin. In two of these, the mgrB gene was truncated by IS10R, while in the third isolate, insertional inactivation of mgrB by ISKPn14 was identified. Hungary is the first European country showing endemic spread of blaDHA-1 facilitated by the international high-risk clone ST11. The rapid countrywide spread of this multidrug-resistant clone seriously endangers Hungarian healthcare facilities and warrants strengthening of infection control practices and prudent use of carbapenems and colistin.

Emergence of KPC-2-producing Escherichia coli isolates in an urban river in Harbin, China.

Three KPC-2-producing Escherichia coli (E1, E2, and E3) were recovered from water samples of an urban river in the city of Harbin, China. Antimicrobial susceptibility was determined by broth microdilution. Molecular characterization and genetic relatedness of the isolates were determined by polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and PCR-directed phylotyping. Plasmids were analyzed by conjugation, S1-PFGE, Southern blotting and PCR-based replicon typing (PBRT). The genetic environment of the bla KPC-2 gene was determined using PCR and sequencing. PCR analyses revealed that the E1 isolate carried the bla KPC-2, bla CMY-2, bla TEM-1, bla CTX-M-14, and qnrB2 genes and belonged to sequence type ST410, phylogenetic type A; the E2 isolate was assigned to ST131-B2 and carried the bla KPC-2, bla TEM-1, bla CTX-M-3, bla DHA-1, aac(6')-Ib-cr, and qnrS1 genes; while the E3 isolate was of ST648-D and possessed bla KPC-2, bla TEM-1, bla OXA-1, bla CTX-M-15, armA, and aac(6')-Ib-cr genes. PFGE demonstrated that each of the three KPC-2-producing E. coli isolates exhibited an individual XbaI patterns. The bla KPC-2 gene was located on plasmids of 60-140 kb with IncA/C, IncN, or non-typeable replicon types. The genetic environment of bla KPC-2 of the three strains was consistent with the genetic structure of bla KPC-2 on the plasmid pKP048.

Virulence Gene Profiles of Multidrug-Resistant Pseudomonas aeruginosa Isolated From Iranian Hospital Infections.

Background:The most common hospital-acquired pathogen is Pseudomonas aeruginosa. It is a multidrug resistant bacterium causing systemic infections.Objectives:The present study was carried out in order to investigate the distribution of virulence factors and antibiotic resistance properties of Pseudomonas aeruginosa isolated from various types of hospital infections in Iran.Patients and Methods:Two-hundred and seventeen human infection specimens were collected from Baqiyatallah and Payambaran hospitals in Tehran, Iran. The clinical samples were cultured immediately and samples positive for P. aeruginosa were analyzed for the presence of antibiotic resistance and bacterial virulence genes using PCR (polymerase chain reaction). Antimicrobial susceptibility testing was performed using disk diffusion methodology with Müeller–Hinton agar.Results:Fifty-eight out of 127 (45.66%) male infection specimens and 44 out of 90 (48.88%) female infection specimens harbored P. aeruginosa. Also, 65% (in male specimens) and 21% (in female specimens) of respiratory system infections were positive for P. aeruginosa, which was a high rate. The genes encoding exoenzyme S (67.64%) and phospholipases C (45.09%) were the most common virulence genes found among the strains. The incidences of various β-lactams encoding genes, including blaTEM, blaSHV, blaOXA, blaCTX-M, blaDHA, and blaVEB were 94.11%, 16.66%, 15.68%, 18.62%, 21.56%, and 17.64%, respectively. The most commonly detected fluoroquinolones encoding gene was gyrA (15. 68%). High resistance levels to penicillin (100%), tetracycline (90.19%), streptomycin (64.70%), and erythromycin (43.13%) were observed too.Conclusions:Our findings should raise awareness about antibiotic resistance in hospitalized patients in Iran. Clinicians should exercise caution in prescribing antibiotics, especially in cases of human infections.