CTX-M Extended-spectrum Beta-lactamases Research Articles

668. Presence of the Narrow-Spectrum OXA-1 Beta-Lactamase Enzyme is not Associated with Elevated Ceftolozane-Tazobactam MIC Values among ESBL-Producing Escherichia coli Clinical Isolates (CANWARD, 2011-2018)

Abstract Background Beta-lactam-beta-lactamase inhibitor combinations have been proposed as an alternative to carbapenems for the treatment of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli as an antimicrobial stewardship initiative. However, in a recent randomized trial evaluating patients with bacteremia, presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Enterobacterales was associated with higher piperacillin-tazobactam MICs, and in turn excess mortality among patients treated with piperacillin-tazobactam relative to meropenem. The purpose of this study was to determine whether the in vitro activity of ceftolozane-tazobactam versus ESBL-producing E. coli is similarly compromised by the presence of OXA-1. Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2011 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection. Results In total, 485 ESBL-producing E. coli identified as part of the CANWARD study were included. The majority of isolates (91.3%; 443/485) harbored a CTX-M ESBL enzyme. OXA-1 was present in 39.6% (192/485) of isolates. OXA-1 was detected in 62.5% (187/299) of isolates with a CTX-M-15 ESBL enzyme versus only 2.7% (5/186) of isolates with other ESBL enzyme types. Ceftolozane-tazobactam MIC50 and MIC90 values were identical (0.25 µg/mL and 1 µg/mL, respectively) for the isolate subsets with and without OXA-1. Overall, 97.4% and 96.9% of isolates with and without OXA-1 remained susceptible (CLSI breakpoint) to ceftolozane-tazobactam. Conclusion The presence of OXA-1 among ESBL-producing E. coli clinical isolates was not associated with an elevation in MIC values for ceftolozane-tazobactam. These data support further evaluation of ceftolozane-tazobactam as an alternative to carbapenems for the treatment of infections caused by ESBL-producing E. coli, as is being done with the MERINO-3 trial. Disclosures George Zhanel, PhD, Merck: Grant/Research Support.

Amplification of the Chromosomal blaCTX-M-14 Gene in Escherichia coli Expanding the Spectrum of Resistance under Antimicrobial Pressure.

ABSTRACTVarious forms of adaptive evolution occur in clinical isolates in response to the presence of antimicrobial drugs. Among a total of 171 CTX-M-9 group/family extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli blood isolates recovered between 2016 and 2017 in six general hospitals, 50.3% of the isolates possessed the blaCTX-M-14-like gene in their chromosome rather than in a plasmid. Focusing on this unprecedented way of the blaCTX-M ESBL gene possession, molecular epidemiology of the isolates was assessed and the chromosomal location of the acquired cephalosporinase gene was dissected in an evolutionary point of view. Taking advantage of a complete collection of E. coli blood isolates from a limited period, clonal relatedness of the E. coli isolates carrying the blaCTX-M-14-like gene was clarified and the dominant clone, ST131 H30R, was identified. To control the level of resistance and the resistance spectrum to oxyimino-cephalosporin drugs, transcription level of the blaCTX-M-14-like gene was tuned finely through positioning the gene near the chromosomal initiation dnaA gene and amplifying numbers of the gene in a chromosome using either the copy-and-paste or the tandem amplification methods. Inconspicuous fitness cost by chromosomal location of the gene and free adjustment of the oxyimino-cephalosporin resistance would urge the dominancy of E. coli clinical isolates harboring the blaCTX-M ESBL gene in their chromosome.IMPORTANCE Increasing prevalence of E. coli producing CTX-M ESBL is a major concern in clinical settings because it significantly limits treatment options. Thus, it is important to keep watching current molecular mechanisms of resistance and the scheme for dissemination. Recently, chromosomal locations of the blaCTX-M genes are often documented in clinical settings and the bacterial strategies were needed to be dissected in an evolutionary point of view. Both main mechanisms of fine tuning the chromosomal gene expression, bacterial gene amplification either by copy-and-paste or by tandem amplification and positioning the gene near the chromosomal initiation dnaA gene, were demonstrated in the study, and the fitness cost by the chromosomal location was evaluated.

Assessment of extended – spectrum beta lactamases (ESBL) gene among gram-negative bacilli bacteria causing community acquired urinary tract infection among females in southwestern Nigeria

Background: Urinary tract infections (UTI) is commoner in females and mainly caused by Gram Negative bacilli. The emergence of Extended – spectrum beta lactamases (ESBL) has made its treatment difficult especially in the community because of limited therapeutic options. This study was undertaken to determine the prevalence of ESBL-producing Gram negative bacilli causing UTI among females in Babcock University Community; evaluate the associated risk factors and to determine the prevalence of the associated genes among the ESBL isolates.
 Methods: A descriptive cross-sectional study of which 200 female participants with clinical UTI were recruited in Babcock University Community. Pretested interviewer–based structured questionnaire was used to obtain the socio-demographic and risk factors. Mid-stream urine was also collected for evaluation of ESBL- producing Gram-negative bacilli. DNA extraction was done for ESBL positive isolates and SHV, TEM, CTX-M and OXA ESBL genes were detected by PCR. SPSS Version 23.0 was used for data analysis.
 Results: The 200-urine samples tested from the participants yielded 33-Gram negative bacilli isolates. Out of these 33 isolates, 17/33(51.5%) were ESBL-producers giving prevalence rate of 8.5% among the participants. The distribution of the ESBL genes among the ESBL isolates was as follows: SHV 12/17(70.6%), TEM 4/17(23.5%), CTX-M 3/17(17.6%) and OXA 5/17(29.4%). No associated risk factors were found.
 Conclusion: All the four types of ESBL genes evaluated were present among the participants. Routine screening for ESBL for Community acquired UTI is necessary to curb treatment failure.

Assessment of extended – spectrum beta lactamases (ESBL) gene among gram-negative bacilli bacteria causing community acquired urinary tract infection among females in southwestern Nigeria

Background: Urinary tract infections (UTI) is commoner in females and mainly caused by Gram Negative bacilli. The emergence of Extended – spectrum beta lactamases (ESBL) has made its treatment difficult especially in the community because of limited therapeutic options. This study was undertaken to determine the prevalence of ESBL-producing Gram negative bacilli causing UTI among females in Babcock University Community; evaluate the associated risk factors and to determine the prevalence of the associated genes among the ESBL isolates.
 Methods: A descriptive cross-sectional study of which 200 female participants with clinical UTI were recruited in Babcock University Community. Pretested interviewer–based structured questionnaire was used to obtain the socio-demographic and risk factors. Mid-stream urine was also collected for evaluation of ESBL- producing Gram-negative bacilli. DNA extraction was done for ESBL positive isolates and SHV, TEM, CTX-M and OXA ESBL genes were detected by PCR. SPSS Version 23.0 was used for data analysis.
 Results: The 200-urine samples tested from the participants yielded 33-Gram negative bacilli isolates. Out of these 33 isolates, 17/33(51.5%) were ESBL-producers giving prevalence rate of 8.5% among the participants. The distribution of the ESBL genes among the ESBL isolates was as follows: SHV 12/17(70.6%), TEM 4/17(23.5%), CTX-M 3/17(17.6%) and OXA 5/17(29.4%). No associated risk factors were found.
 Conclusion: All the four types of ESBL genes evaluated were present among the participants. Routine screening for ESBL for Community acquired UTI is necessary to curb treatment failure.

Phenotypic and Molecular Characterization of CTX-M Type B-Lactamases in Gram Negative Bacterial Strains Isolated from Hospitals, Lahore, Pakistan.

One of the principal mechanisms that contribute resistance to antibiotics is the production of extended spectrum beta lactamase (ESBL) in Gram negative bacteria. In the present study, molecular methods were used to evaluate the prevalence of the extended-spectrum beta-lactamase (ESBL)-encoding CTX-M gene among Gram negative bacterial strains. In total, 148 clinical samples were collected from different tertiary care hospitals of Lahore, Pakistan. Disc synergy diffusion method was used to detect the presence of ESBL production. Moreover, antibiotic resistance patterns and molecular detection of bla CTX-M ESBLs, were also studied. The pathogens isolated from the 148 samples included Escherichia coli (43%) followed by Klebsiella sp. (28%), Proteus sp. (18%) and Pseudomonas sp. (11%). In all 148 strains, 95 (64%) were ESBL producers while 53 (36%) were non ESBL producers. The strains which were phenotypically ESBL producers, bla CTX-M were found in 46% E. coli strains, while 50% Klebsiella sp. were harboring the gene. A high resistance rate was observed against cephalosporins (cefopodoxime 67%, cefoperazone 73%, cephalexin 63% sparaxin 61%). Lower resistance was observed against meropenem among all isolated bacterial strains. Genotypic detection of bla CTX-M genes by PCR revealed 46% of E. coli and 50% of Klebsiella strains harbored bla CTX-M gene. The present study showed that ESBLs producers were resistant to commonly used antibiotics. Similarly, bla CTX-M ESBL production is more prevalent in our clinical isolates.

1216. Presence of the Narrow-Spectrum OXA-1 Beta-lactamase Enzyme Is Associated with Elevated Piperacillin-Tazobactam MIC Values Among ESBL-producing Escherichia coli Clinical Isolates (CANWARD, 2007-2018)

BackgroundThe clinical outcome of patients with bacteremia due to an extended-spectrum beta-lactamase (ESBL)-producing member of the family Enterobacteriaceae who are treated with piperacillin-tazobactam appears to depend, at least in part, on the piperacillin-tazobactam MIC. The purpose of this study was to determine whether there is any association between the MIC of piperacillin-tazobactam and presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Escherichia coli.Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2007 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection.ResultsIn total, 671 ESBL-producing E. coli were identified as part of the CANWARD study. The majority of isolates (92.0%; 617/671) harbored a CTX-M ESBL enzyme. CTX-M-15 (62.3%; 418/671), CTX-M-27 (13.9%; 93/671), and CTX-M-14 (13.4%; 90/671) were the most common variants identified. The narrow spectrum OXA-1 beta-lactamase enzyme was present in 42.6% (286/671) of isolates. OXA-1 was detected in 66.3% (277/418) of isolates with a CTX-M-15 ESBL enzyme versus only 3.6% (9/253) of isolates with other ESBL enzyme types. The piperacillin-tazobactam MIC50 and MIC90 values were 8 µg/mL and 32 µg/mL for isolates that possessed the OXA-1 enzyme versus 2 μg/mL and 8 µg/mL for those that did not. The percentage of ESBL-producing E. coli isolates that were inhibited by a piperacillin-tazobactam MIC of ≤8 μg/mL was 68.5% for isolates that were OXA-1 positive and 93.8% for isolates that were OXA-1 negative.ConclusionThe MIC50 and MIC90 values of piperacillin-tazobactam among ESBL-producing E. coli were higher for the subset of isolates that harbored a narrow spectrum OXA-1 beta-lactamase enzyme relative to the subset that did not. This association was primarily observed among ESBL-producers with the CTX-M-15 enzyme variant. OXA-1 was infrequently detected among isolates with other ESBL enzyme types.Disclosures George Zhanel, PhD, AVIR (Grant/Research Support)Iterum (Grant/Research Support)Merck (Grant/Research Support)Pfizer (Grant/Research Support)Sandoz (Grant/Research Support)Sunovion (Grant/Research Support)Venatorx (Grant/Research Support)Verity (Grant/Research Support)

Detection of CTX-M-Type Extended-Spectrum Beta-Lactamase Producing Salmonella Typhimurium in Commercial Poultry Farms in Copperbelt Province, Zambia

In Zambia, poultry is a rapidly increasing sector contributing 4.8% of the Agricultural Gross Domestic Product (GDP), thus providing a significant income-generating activity. Worldwide, poultry is a major reservoir of Salmonella with an increasing incidence of extended-spectrum beta-lactamase (ESBL) producing strains. ESBLs are enzymes produced by bacteria and are capable of inactivating a wide range of beta-lactam antibiotics. Salmonella enterica serovars Enteritidis and Typhimurium are the most important foodborne serotypes in many countries, infecting both humans and animals and are transmitted to humans through the food supply chain. CTX-M ESBLs have been described in Salmonella Typhimurium isolates with resistant genes located on transferable plasmids. This study aimed to detect S. Typhimurium, their antimicrobial resistance, and CTX-M-type ESBL Producing strains in commercial poultry farms in Copperbelt Province, Zambia. Five districts were considered for this study, where one poultry farm per district was randomly selected for sampling. An overall number of 384 fecal samples were analyzed using microbiological and molecular methods. S. Typhimurium was detected at 17.7% (CI: 14.2%-21.8%) in commercial poultry farms in Copperbelt Province, of which 12.8% (CI: 9.8%-16.5%) were found harboring the CTX-M-Type ESBL genes. S. Typhimurium isolates showed 88.2% resistance to at least one antimicrobial compound. All the isolates showed 100% resistance to tetracycline, followed by ampicillin and amoxicillin at 91.2%. These isolates also showed 58.8% resistance to cefotaxime and 54.4% to ceftazidime. Detection of CTX-M ESBL producing Salmonella Typhimurium suggests the contamination of chicken food chain at farm level and calls for public health protection measures.

Detection of international high-risk clones of food-borne pathogens Salmonella and Escherichia coli in the Russian Federation

The use of whole genome sequencing, standard analyze methods and international databases allows to detect international high-risk clones of food-borne pathogens, to assess their evolution and geographical distribution due to international trade of animal food products and farm animals. In Saint-Petersburg, Russian Federation, Salmonella Newport, Salmonella Kentucky and Escherichia coli O26:H11 of well-known international high-risk clones were first identified from patients with diarrhea. Two S. Kentucky strains isolated in 2015 and 2019 were identical to the international clone ST198 widely distributed in European countries: had multidrug resistance, high-level fluoroquinolone resistance (MIC of ciprofloxacin 32,0 mg/l) due to three single-nucleotide substitutions in the gyrA (Ser83Phe and Asp87Asn) and parC (Ser80Ile). In 2008, a multidrug resistant S. Newport strain resistant to extended-spectrum cephalosporins due to AmpC-cephalosporinase CMY-2 was isolated, belonging to the international clone of S. Newport MDR-AmpC/CMY-2 which caused sporadic cases and outbreaks in the USA and Europe in the 2000s. The plasmid containing the blaCMY-2 had a PstI-restriction profile, one of the most prevalent in the international clone. E. coli O26:H11 strains, isolated in Saint-Petersburg, produced the shiga-like toxin STX1 (stx1a), had additional virulence genes: ehxA (enterohemolysin), katP (catalase peroxidase), espP (serine protease), as well as cba (colicin B), gad (glutamate decarboxylase), cif (type III secreted effector), iss (increased serum survival), belonged to the phylogenetic group B1 and the international high-risk clone E. coli O26:H11 ST21 widely distributed in Europe and the USA. 25% of the strains had multidrug resistance and produced CTX-M extended spectrum beta-lactamases. In the Russian Federation E. coli O26 is consider as the pathogen of diarrheal diseases and registered as Enteropathogenic E. coli in routine bacteriological laboratories without detecting of H-antigen and shiga-like toxins.

Molecular characterization of bla TEM and bla CTX-M in Multi-Drug Resistance E. coli isolated from Country Chicken and Country Chicken Eggs in Poultry Farms of Malaysia

Over the years, multidrug-resistant Escherichia coli has contributed to the development of extended-spectrum beta-lactamase (ESBLs), which evolved primarily from poultry in every corner of the world. The unregulated use of antibiotics commonly administered to poultry products to prevent any subclinical infections that lead to multidrug resistance (MDR) that is due to acquired bacteria resistance. The main aim of this study is to investigate the prevalence in Malaysia of multidrug-resistant, extended-spectrum beta-lactamase (ESBL) producing E.coli from poultry country chicken and country eggs. In several states of Malaysia, fifty samples from country chicken and country chicken eggs were extracted randomly from chosen poultry. The sample was taken from May to June 2019. The samples were tested using traditional microbiological techniques for the presence of E. coli. Antibiotics susceptibility test using 5 forms of β-lactam antibiotics was used using the double-disk diffusion screening, and confirmation of the test is performed by a combination disk diffusion process to establish the strains generating the ESBL. Although, the phenotypic characterization of bla TEM and bla CTX-M ESBL was carried out using PCR and SDS methods. Twenty of the fifty samples collected were classified as E.coli (20/50), suggesting 40%. The results of the distribution of the β-lactamases genes were reported as bla TEM, bla CTX-M with results of 75% (3/4), and 100% (4/4), respectively. The findings indicate a high prevalence of multidrug resistance as the most prevalent of all ESBL genes in ESBL infections with CTX-M genes. Consequently, effective monitoring of MDR infections, in particular resistance to β-lactamases in poultry chicken, can predict the potential for ESBL infections in humans and animals.

A highly multiplexed melt-curve assay for detecting the most prevalent carbapenemase, ESBL, and AmpC genes

Resistance to third-generation cephalosporins and carbapenems in Gram-negative bacteria is chiefly mediated by beta-lactamases including extended-spectrum beta-lactamase (ESBL), AmpC, and carbapenemase enzymes. Routine phenotypic detection methods do not provide timely results, and there is a lack of comprehensive molecular panels covering all important markers. An ESBL/carbapenemase high-resolution melt analysis (HRM) assay (SHV, TEM, CTX-M ESBL families, and NDM, IMP, KPC, VIM and OXA-48–like carbapenemases) and an AmpC HRM assay (16S rDNA control, FOX, MOX, ACC, EBC, CIT, and DHA) were designed and evaluated on 111 Gram-negative isolates with mixed resistance patterns. The sensitivity for carbapenemase, ESBL, and AmpC genes was 96.7% (95% confidence interval [CI]: 82.8–99.9%), 93.6% (95% CI: 85.7–97.9%), and 93.8% (95% CI: 82.8–98.7%), respectively, with a specificity of 100% (95% CI: 95.6–100%), 93.9% (95% CI: 79.8–99.3%), and 93.7% (95% CI: 84.5–98.2%). The HRM assays enable the simultaneous detection of the 14 most important ESBL, carbapenemase, and AmpC genes and could be used as a molecular surveillance tool or to hasten detection of antimicrobial resistance for treatment management.

Emergence of diversity in carbapenemase-producing Escherichia coli ST131, England, January 2014 to June 2016.

Background Escherichia coli ST131, a global, high-risk clone, comprises fluoroquinolone resistance (FQ-R) mutations and CTX-M extended-spectrum beta-lactamases associated with the fimH30-encoding clades, C1 and C2. Further carbapenem resistance development in ST131 is a public health concern.AimThis observational study aimed to probe the diversity of carbapenemase-producing E. coli (CP E. coli) ST131 across England.MethodsST131 isolates were identified using whole-genome sequencing (WGS) data generated for all non-duplicate CP E. coli from human samples submitted to the national reference laboratory from January 2014 to June 2016. Antimicrobial resistance (AMR) gene content and single nucleotide polymorphism (SNP) data were compared against a published ST131 phylogeny and analysed alongside patient metadata.ResultsThirty-nine genetically diverse ST131 CP E. coli, from eight of nine regions, represented 10% of CP E. coli isolates sequenced. Ten and eight isolates were from the FQ-susceptible (FQ-S) clades A and B, while eight and 15 isolates belonged to the FQ-R clades C1 or C2, respectively. Seven distinct carbapenemases were identified: KPC-2 (21 isolates, 6 regions) frequently occurred among clade C2 isolates (n = 10). OXA-48-producers (10 isolates, 3 regions) were often from clade A (n = 5). NDM-1 (n = 4), NDM-5 (n = 1), VIM-1 (n = 1), VIM-4 (n = 1) and OXA-181 (n = 1) were also identified. Clade C2 isolates encoded more AMR genes than those from clades A (p = 0.02), B (p = 9.6 x 10−3) or C1 (p = 0.03).ConclusionWhen compared with its global predominance among ESBL-E. coli, ST131 represented a fraction of the CP E. coli received, belonging to diverse clades and encoding diverse carbapenemases. The greater accumulation of resistance genes in clade C2 isolates highlights the need for ongoing monitoring of this high-risk lineage.

Identifying the mechanism underlying treatment failure for Salmonella Paratyphi A infection using next-generation sequencing \u2013 a case report

BackgroundSalmonella is a notorious pathogen that causes gastroenteritis in humans and the emergence of resistance to third-generation cephalosporins and azithromycin have raised concern. There has been rare case of Salmonella Paratyphi A infection accompanied by spondylitis. Here, we report a case of initial antibiotic treatment failure in a Korean man with Salmonella Paratyphi A infection and conducted next-generation sequencing (NGS) to determine the cause of failure of initial treatment for Salmonella Paratyphi A infection.Case presentationA 70-year-old man was admitted to Chosun University Hospital with reported consistent low back pain with a history of having 5 days of chills and fever in another hospital a month ago. He was administered ceftriaxone (2 g daily) for 18 days including initial treatment to cover Salmonella enterica. The antimicrobial susceptibility test using MIC plate, found that the identified organism was resistant to ciprofloxacin and nalidixic acid. Moreover, the Salmonella Paratyphi A isolates were found to have an MIC > 16 mg/L for azithromycin, as he had resistance to both azithromycin and nalidixic acid, the treatment was switched to a combination of ciprofloxacin and cefotaxime. We carried out next-generation sequencing (NGS) to determine the cause of failure of initial treatment for Salmonella Paratyphi A infection. NGS showed that the amino acid substitution GyrA S83F and the expression of multiple RNA-family efflux pumps led to a high-level resistance to quinolone. No genes related to ceftriaxone resistance, such as CTX-M, CMY-2, or other extended-spectrum beta-lactamases were identified in Salmonella enterica Paratyphi A using NGS. The GyrA S83F mutation and the expression of multiple RNA-family efflux pumps may have contributed to the treatment failure of ceftriaxone, even though the MIC of the isolate to ceftriaxone was less than 1.ConclusionThis case involved a Salmonella Paratyphi A infection accompanied by spondylitis. To our knowledge, this is the first report to elucidate the mechanism underlying antimicrobial resistance using NGS.

Acquisition and Loss of CTX-M-Producing and Non-Producing Escherichia coli in the Fecal Microbiome of Travelers to South Asia.

Over 80% of travelers from the United Kingdom to the Indian subcontinent acquire CTX-M-producing Escherichia coli (CTX-M-EC), but the mechanism of CTX-M-EC acquisition is poorly understood. We aimed to investigate the dynamics of CTX-M-EC acquisition in healthy travelers and how this relates to populations of non-CTX-M-EC in the fecal microbiome. This is a prospective observational study of healthy volunteers traveling from the United Kingdom to South Asia. Fecal samples were collected pre- and post-travel at several time points up to 12 months post-travel. A toothpicking experiment was used to determine the proportion of cephalosporin-sensitive E. coli in fecal samples containing CTX-M-EC. MLST and SNP type of pre-travel and post-travel E. coli were deduced by WGS. CTX-M-EC was acquired by 89% (16/18) of volunteers. Polyclonal acquisition of CTX-M-EC was seen in 8/15 volunteers (all had >3 STs across post-travel samples), suggesting multiple acquisition events. Indistinguishable CTX-M-EC clones (zero SNPs apart) are detectable in serial fecal samples up to 7 months after travel, indicating stable maintenance in the fecal microbiome on return to the United Kingdom in the absence of selective pressure. CTX-M-EC-containing samples were often co-colonized with novel, non-CTX-M strains after travel, indicating that acquisition of non-CTX-M-EC occurs alongside CTX-M-EC. The same pre-travel non-CTX-M strains (<10 SNPs apart) were found in post-travel fecal samples after CTX-M-EC had been lost, suggesting return of the fecal microbiome to the pre-travel state and long-term persistence of minority strains in travelers who acquire CTX-M-EC.IMPORTANCEEscherichia coli strains which produce CTX-M extended-spectrum beta-lactamases are endemic as colonizers of humans and in the environment in South Asia. This study demonstrates that acquisition of CTX-M-producing E. coli (CTX-M-EC) in travelers from the United Kingdom to South Asia is polyclonal, which is likely due to multiple acquisition events from contaminated food and drinking water during travel. CTX-M-EC frequently persists in the fecal microbiome for at least 1 year after acquisition, often alongside newly acquired non-CTX-M E. coli strains. In travelers who acquire CTX-M-EC, pre-travel non-CTX-M E. coli remains as a minority population in the gut until the CTX-M-EC strains are lost. The non-CTX-M strains are then reestablished as the predominant E. coli population. This study has shed light on the dynamics of CTX-M-EC acquisition, colonization, and loss after travel. Future work involving manipulation of nonvirulent resident E. coli could be used to prevent colonization with antibiotic-resistant E. coli.

Impact of host-pathogen-treatment tripartite components on early mortality of patients with Escherichia coli bloodstream infection: Prospective observational study.

BackgroundRisk factors affecting early morality of patients with Escherichia coli bloodstream infection (BSI) were investigated including the host-pathogen-treatment tripartite components.MethodsSix general hospitals in South Korea participated in this multicentre prospective observational study from May 2016 to April 2017 and a total of 1492 laboratory-confirmed E. coli BSI cases were studied. Cox regression was used to estimate risks of the primary endpoint, i.e., all-cause mortality within 30 days from the initial blood culture. Six multivariate analysis models were constructed in accordance to the clinical importance and intra- and inter-component multicollinearity.FindingsAmong the 1492 E. coli BSI cases, 9.5% (n = 141) patients expired within 30 days. Six models of multivariate analysis indicated risk factors of critical illness, primary infection of peritoneum, and chronic liver disease including cirrhosis for host variables; of phylogenetic group B2, ST131-sublineage H30Rx, multidrug resistance, group 1 CTX-M extended-spectrum beta-lactamase production, and having either of fyuA, afa, and sfa/foc virulence genes for causative E. coli pathogen variables; and of delayed definitive therapy for antimicrobial treatment variables. In addition, as a protective factor, primary urinary tract infection was identified.InterpretationDespite decades' effort searching for the risk factors for E. coli BSI, systemic understanding covering the entire tripartite component is still lacking. This study detailed the organic impact of host-pathogen-treatment tripartite components for early mortality in patients with E. coli BSI.

The frequency of Klebsiella pneumonia encoding genes for CTX-M, TEM-1 and SHV-1 extended-spectrum beta lactamases enzymes isolated from urinary tract infection

BackgroundThe extended- spectrum β-lactamase producing bacteria are widely spread worldwide. The productions of these enzymes cause bacterial resistance to a wide range of antibiotics. The aim of this study was to investigated the frequency of K. pneumonia encoding genes for CTX-M, TEM-1 and SHV-1 extended-spectrum beta lactamases enzymes isolated from urinary tract infection.MethodsThis study is cross-sectional study. All K. pneumonia isolates from urine samples, which had grown on media culture more than 105 were delivered to the medical microbiology laboratory. K. pneumonia susceptibility of 198 samples were confirmed by disk diffusion. The gene frequency of genes was determined using PCR, and analyzed using SPSS version 21 software.FindingMost of the K. pneumonia isolated from urine producing β-lactamase were resistant to cotrimoxazole (53.2%) followed by cefotaxime (50%), ceftazidime, ceftriaxone (40.3%), nalidixic acid (17.8%), amikacin and imipenem (1.6%) and meropenem (0%) respectively. Out of the 198 confirmed isolates of K. pneumonia, 62 cases (31.3%) have the gene phenotype of broad spectrum β-lactamase enzymes and highest frequency of gene phenotype was related to the SHV-1 gene (85.5%). Then in the terms of abundance from highest to lowest CTXM-3 (56.5%), CTXM-1 (27.4%), TEM-1 (16.1%) and CTXM-2 (8.1%), were respectively.ConclusionThis study showed that K. pneumonia isolated from urine producing β-lactamase were resistance to a wide range of antibiotics. Due to the increasing resistance of most antibiotics, control and supervision in the use of antibiotics and identification of broad spectrum β-lactamase enzymes by phenotypic methods appears to be essential.

SHV and CTX-M Extended Spectrum Beta Lactamases (ESBL) Producing Bacteria Isolated from Street Foods in and around Chennai, India

Street food vending in India has flourished into a prominent trade especially in the urban and semi urban regions of the country. It is also a major reservoir for the spread of food borne infections caused by microbial pathogens including multidrug resistant organisms. Management of infections caused by multidrug resistant bacteria is difficult. Extended spectrum beta lactamase (ESBL) production contribute to antimicrobial resistance that may result in therapeutic failure. The objective of this study was to analyze for the prevalence of ESBL producing food borne bacteria isolated from street foods sold in and around Chennai. A total of 78 street foods including vegetable samosa (18), chicken samosa (10), panipuri masala (11), panipuri water (31), bhelpuri (8) were tested for the presence of ESBL producing bacteria. The bacteria that were identified included Escherichia coli (55.1%), Staphylococcus sp. (25.6%), Klebsiella sp. (17.9%), Pseudomonas sp. (15.3%) and Enterobacter sp. (11.5%). Multidrug resistance was exhibited by the isolates and ESBL production was found in 16 out of 78 (20.5%) isolates with 9 E. coli, 2 Klebsiella sp., and 5 Pseudomonas sp. Polymerase Chain Reaction (PCR) revealed CTX-M-1(68.75%) and SHV genotypes (31.25%) among the isolates. The 16SrRNA analysis of F10 isolate, confirmed the presence of Pseudomonas sp. in street foods.

Molecular Characterization of CTX-M Producing Salmonella Isolates with Concurrent Resistance to Ciprofloxacin and Cefotaxime from Slaughtered Chicken Carcasses in Qingdao, China

Objectives: To describe the molecular characterization of CTX-M ESBL (Extended-spectrum Beta-lactamase)- producing Salmonella co-resistant ciprofloxacin and cefotaxime from chicken carcasses in 2014 in Qingdao, China. Methods: A total of 62 Salmonella isolates were confirmed as ESBLs by a combination disc diffusion test from 355 Salmonella isolates studied. Antimicrobial susceptibility was determined by the broth dilution method. CTX-M subtypes, Quinolone Resistance-determining Regions (QRDRs), Plasmid-mediated Quinolone Resistance (PMQR) determinants and Multi-locus Sequence Typing (MLST) were identified using PCR (Polymerase Chain Reaction) and sequencing. Plasmids were characterized by S1-nuclease PFGE, PCR-based replicon typing and transformation. Broth mating assays were carried out for all isolates to determine whether the CTX-M or PMQR marker could be transferred by conjugation. Results: Of the 62 ESBL-producing Salmonella isolates, showing resistant to a multiple antimicrobials, 59 isolates were identified as S. indiana (ST17) followed by two S. enteritidis and one S. typhimurium. Moreover, all 62 ESBLs were subtyped to six blaCTX-M types, with blaCTX-M-123 being the most predominant, followed by blaCTX-M-65, blaCTX-M-130, blaCTX-M-14, blaCTX-M-24, and blaCTX-M-79. Amino acid substitutions in GyrA (S83F and D87N) and ParC (T57S and S80R) and the PMQR-encoding genes [aac(6′)-Ib-cr, oqxA, oqxB, qnrB, and qnrS] were detected in almost all tested isolates. All isolates contained one to three large plasmids and 8 replicon types were detected with IncHI1, IncHI2, IncA/C, IncFIIAS, and IncFrep replicon types being the predominating incompatibility groups. Twenty-six isolates demonstrated the ability to transfer their CTX-M and PMQR marker at different transfer rates. Conclusion: The emergence of CTX-M, QRDR and PMQR were found in ciprofloxacin and cefotaxime coresistant Salmonella, especially S. indiana among chicken carcasses in Qingdao. To the best of our knowledge, this is the first report of blaCTX-M-123 and blaCTX-M-130 in Salmonella. The high prevalence and conjugative characteristics suggest that control strategies are necessary to limit the dissemination of these isolates through food chain.

Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics

Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics.

Prevalence of MDR pathogens of bacterial meningitis in Egypt and new synergistic antibiotic combinations.

The aim of this study was identifying bacterial pathogens involved in meningitis, studying their antibiotic resistance profiles, investigating the antibiotic resistance genes as well as evaluating the use of various antibiotic combinations. Antibiotic susceptibility tests were evaluated according to CLSI guidelines. Antibiotic combinations were evaluated by calculating the Fractional Inhibitory Concentration (FIC) index. A total of 71 bacterial isolates were recovered from 68 culture positive CSF specimens. Sixty five of these isolates (91.5%) were recovered from single infection specimens, while 6 isolates (8.4%) were recovered from mixed infection specimens. Out of the 71 recovered isolates, 48 (67.6%) were Gram-positive, and 23 (32.4%) were Gram-negative. Thirty one of the Gram positive isolates were S. pneumoniae (64.6%, n = 48). Out of the recovered 71 isolates; 26 (36.6%) were multidrug-resistant (MDR) isolates of which, 18 (69.2%) were Gram-negative and 8 (30.8%) were Gram-positive. All MDR isolates (100%) showed resistance to penicillin and ampicillin, however, they showed lower resistance to meropenem (50%), levofloxacin (50%), amikacin (48%), pipercillin-tazobactam (45.8%). Most common antibiotic resistance genes were investigated including: tem (21.1%), shv (15.8%), ctx-m (15.8%) coding for TEM-, SHV, CTX-M extended-spectrum beta-lactamases (ESBLs), respectively; aac(6')-I b(26.3%) coding for aminoglycoside 6’-N-acetyltransferase type Ib ciprofloxacin resistant variant; and qnrA (5.3%) gene coding for quinolone resistance. The DNA sequences of the respective resistance genes of some selected isolates were PCR amplified, analyzed and submitted to the GenBank database under the accession numbers, KX214665, KX214664, KX214663, KX214662, respectively. The FIC values for ampicillin/sulbactam plus cefepime showed either additive or synergistic effect against ten tested Gram-negative MDR isolates, while doxycycline plus levofloxacin combination revealed synergism against two MDR Gram-positive isolates. The results indicate high prevalence of antibiotic resistance among MDR isolates. Therefore, new guidelines should be implemented in Egypt to rationalize the use and avoid the misuse and abuse of antimicrobial agents.

HIGHER CTX-M, TEM, AND SHV EXTENDED-SPECTRUM BETA-LACTAMASE PLASMID GENE COMBINATION FREQUENCY IN ESBL PRODUCING KLEBSIELLA PNEUMONIAE COMPARED WITH ESBL PRODUCING ESCHERICHIA COLI

Introduction:Extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae confer antibiotic resistance to broad-spectrum penicillins, cephalosporins, using ESBL genes CTX-M, TEM, SHV, which are encoded in bacterial plasmid genome. Methods: We compared plasmid CTX-M, TEM, and SHV ESBL gene combinations in ESBL producing Escherichia coliand Klebsiella pneumoniae. Altogether, 136 ESBL producing Enterobacteriaceae isolation cases were analyzed. Results:The ESBL producing Enterobacteriaceae, mostly K. pneumoniae (n = 66; 48.53%), E. coli (n = 36; 26.47%), were isolated from 52 (38.2%) female and 84 (61.8%) male patients from Riga East Clinical University Hospital. Overall,132 isolates (97.06%) registered positive for the plasmid CTX-M gene, 97 isolates (71.32%) for the plasmid TEM gene, and 87 isolates (63.97%) for the plasmid SHV gene.Patients with the three ESBL plasmid gene (CTX-M+ TEM+ SHV+) combination were significantly older (67.11 ± 14.16 years) than patients with other gene combinations (59.63 ± 18.14 years; p = 0.047). Plasmid SHV gene frequency was higher in K. pneumoniae (p &lt; 0.001). The K. pneumoniae mostly presented with the three plasmid ESBL gene (CTX-M+ TEM+ SHV+) combination, whereas E. coli presented with other gene combinations (p = 0.014). Conclusions: Klebsiella Pneumoniae, more often, presented with the plasmid SHV ESBL gene and the three ESBL gene (CTX-M+ TEM+ SHV+) combination, compared to E. coli. Older patients with ESBL producing Enterobacteriaceae infection, more often, presented with the three ESBL gene (CTX-M+ TEM+ SHV+) combination, compared to younger patients with the infection.