AmpC Production Research Articles

Screening for Extended - Spectrum β-Lactamases, AmpC and Carbapenemases Producing Bacteria among Adult and Pediatric Intensive Care Unit Patients in Two University Hospitals in Egypt

Background: Emerging β-lactam resistant gram-negative bacteria (BLR-GNB) is a problem worldwide, particularly in the Intensive Care Units (ICUs). Objective: The aim of the present work was to evaluate the rate of carriage and the acquisition rate of BLR-GNB at one adult and one pediatric ICU in Alexandria University Hospitals, Egypt. Methodology: One hundred adult and 50 pediatric intensive care unit patients were included in the study. Rectal and nasal swabs were inoculated onto chromID ESBL (BioMerieux). The rate of carriage and the acquisition rate of BLR-GNB were calculated in both ICUs. All identified GNB were tested for ESBL, AmpC-β lactamase and carbapenemase production using combined disc, AmpC disc, and modified Hodge test (MHT) respectively. Results: Eighty eight percent of adult and 74% of pediatric patients were BLR-GNB carriers. The acquisition rate of BLR-GNB in adult & pediatric ICUs 48 hrs after admission was 42.9% and 35% respectively. Out of the 150-screened patients, 280 BLR-GNB isolates were identified where E. coli was the most commonly isolated (45%) followed by K. pneumoniae (35%). Conclusions: The high rate of BLR-GNB carriage calls for routine surveillance, which will be helpful for monitoring the spread of drug resistance in ICU settings.

Molecular epidemiology of beta-lactamase producing nosocomial Gram-negative pathogens from North and South Indian hospitals.

Resistant Gram-negative bacterial (GNB) infections, apart from tremendously escalating the cost of treatment, are a cause for substantial morbidity and mortality among hospitalized patients. Such bacteria are rapidly acquiring resistance to many antimicrobial agents, especially the beta-lactams which are the most frequently prescribed antimicrobials in hospital and community patient care settings, and now also to colistin; a last-line drug to treat infections with such bacteria. The greatest threat to antimicrobial treatment is the production of metallo beta-lactamases, and plasmid-mediated serine carbapenemases. We conducted a two-year study to observe the pattern of beta-lactamase enzyme production (extended spectrum beta-lactamases (ESBLs), AmpC and carbapenemases) among the nosocomial GNB isolated from intensive care units (ICUs) of North and South Indian hospitals. A total of 761 non-duplicate GNB were included in the study from North (554; 73 %) and South India (207; 27 %). All strains were subjected to Clinical and Laboratory Standards Institute (CLSI) recommended screening tests for detection of beta-lactamase production, followed by polymerase chain reaction (PCR)-based detection of clinically important beta-lactamase genes mediating resistant phenotypes among these isolates. Out of the 761 GNB, Acinetobacter spp., Klebsiella spp., Pseudomonas spp., Enterobacter spp. and others were 27, 23 , 21 , 17 , 5 and 7 % respectively. A high prevalence of ESBL was found across all genera in these strains. The carbapenem resistance was higher in North than in South Indian GNB. The level of AmpC production was comparatively lower in both North and South Indian strains. Beta-lactamases showed tremendous variation in geographic distribution. Thus, their detection and characterization is important from a clinical-epidemiological, laboratory and infection control point of view. Knowledge of this epidemiology can predict the empiric antimicrobial treatment.

In vitro susceptibility and resistance phenotypes in contemporary Citrobacter isolates in a University Hospital in Crete, Greece

Background: Data on Citrobacter spp. susceptibility are scarce. We sought to study the evolution in the susceptibility of 385 Citrobacter spp. at the University Hospital of Heraklion, Crete, Greece during a six-year period (2010–2015).Methods: Non-duplicate strains isolated from inpatients (intensive care unit, oncology, surgery, internal medicine, paediatrics) and outpatients were studied using Vitek 2. Phenotypic confirmatory tests were applied for detection of β-lactamases and aminoglycoside modifying enzymes.Results: C. freundii (172, 44.7%) and C. koseri (166, 43.1%) were the most commonly isolated species. C. braakii (34), C. amalonaticus (6), C. youngae (6) and C. sedlakii (1) were the remaining isolates. Colistin and fosfomycin were the most active antibiotics (both 99.2%) followed by carbapenems (99%) aminoglycosides (96.6–98.4%), tigecycline (96.1%), cefepime (94.8%), ciprofloxacin (94.3%), tetracycline (92.7%), trimethoprim/sulphamethoxazole (91.4%), chloramphenicol (88.1%), piperacillin/tazobactam (86.5%) and 3rd generation cephalosporins (85.7%). C. freundii were more resistant than C. koseri. Antibiotic resistance did not increase during the study period for most antibiotics. Lower susceptibility to all antibiotics was observed among multi-drug resistant (MDR) strains. AmpC was the most common resistant mechanism (10.9%); carbapenemases (1.3%) and aminoglycoside modifying enzymes (2.9%) were also detected. All AmpC producers were resistant to cephalosporins but not to carbapenems. In all but one isolates aminoglycoside resistance was accompanied by acquired β-lactamases.Conclusions: Although Citrobacter species in general were susceptible, antibiotic susceptibility testing is required for the detection of resistant isolates.

CTX-M-15 and OXA-10 beta lactamases in multi drug resistant Pseudomonas aeruginosa: First report from Pakistan.

Pseudomonas aeruginosa is an emerging threat to public health worldwide due to their rapid development of drug resistance including beta-lactamases. The present study was designed to investigate the incidence of β-lactamases and genotypic pattern of CTX and OXA in the clinical isolate of multidrug resistant P.aeruginosa. In this study a total of 102 MDR P.aeruginosa isolates obtained from Lady Reading Hospital, Peshawar, Pakistan were subjected to extended spectrum beta lactamase (ESBL), metallo beta lactamase (MBL) and plasmid mediated β-lactamase (AmpC) detection using phenotypic and molecular methods. Furthermore, sequencing of CTX and OXA gene was performed. Out of 102 MDR P.aeruginosa isolates, 71 (69.6%) were beta lactamase producers. The incidence of ESBL, MBL and AmpC in clinical isolates of P.aeruginosa was found to be 23.94%, 40.84% and 35.21% respectively. Co-production of ESBL and AmpC were also observed in some isolates. There were 14 (19.71%) CTX-M-15 harboring isolates which were ESBL (64.28%), MBL (21.42%) and AmpC (14.28%) producer. Co-production of ESBL/MBL (14.28%), ESBL/AmpC (14.28%) and MBL/AmpC (14.28%) were also observed in the CTX M-15 harboring isolates while 12.28% isolates were not ESBL, MBL or AmpC producer. OXA-10 was detected in 8 (11.26%) isolates which were ESBL (12.5%), MBL (37.5%) and AmpC (12.5%) producer. OXA 10 isolates also exhibit co-production of ESBL/AmpC (12.5%) and MBL/AmpC (12.5%). All CTX-M-15 carried the class A β-lactamase conserved domain while OXA-10 harbored conserved domain of class D β-lactamase. The current study for the first time reported and characterized the CTX-M-15 and OXA-10 among MDR P.aeruginosa isolates from Pakistan. Further efforts are needed to understand the molecular mechanism of drug resistance with CTX and OXA harboring P.aeruginosa isolates.

English

Acquisition of multidrug resistance and the spread of MDR among pathogenic microorganisms is a growing concern to world. Extended-spectrum -lactamases (ESBLs) strains of the Enterobacteriaceae; ESBL trait expressing Escherichia coli poses major threat and ESBL E. coli infections are increasingly reported from hospital settings as well as community settings with rising mortality and morbidity rates. The objective of this study was to study distribution of various types of -lactamases expressed within the clinical isolates of E. coli. In this work, clinical specimens: urine, stool, sputum, pus were collected over a period of three months from patients from rural area of Maharashatra State, India. Out of number of isolates, only thirty-six E. coli isolates were chosen for determining existence of ESBL, MBL, AmpC and Carbapenamase producing ability. Choice of isolates included resistance to at least of the three third generation cephalosprins used in this study.  About 75% of E. coli strains were found to express ESBL trait, while remaining 25% expressed non-ESBL trait.  About 28% isolates produced AmpC phenotype. Seven of 36 isolates (18%) indicated co-existence of ESBL and AmpC, while strain AKA43 was found to contain MBL and AmpC traits together.  The prevalence of ESBL, MBL, AmpC and carbapenemase producing E. coli was studied.  A few of these isolates were found to harbor more than one type of -lactamases. Key words: Extended spectrum ß-lactamases (ESBLs), ampCß-lactamases, metallo-ß-lactamases (MBLs), antibiotic resistance.

Fecal carriage of MDROs in a population of Lebanese elderly: Dynamics and impact on bacterial fitness.

Muti-Drug Resistant Organisms (MDROs) are problematic all over the world, especially in Lebanon. High fecal carriage rates of MDR Enterobacteriaceae were reported from Lebanese nursing homes. Some studies show that MDROs have a fitness cost as compared to sensitive isolates. In this study, the competitive growth of MDR Escherichia coli obtained from fecal samples from elderly is assessed. Fecal swabs from ten elderly patients from a Lebanese nursing home were obtained between June and December, 2015. Isolates were identified by API 20E and antimicrobial susceptibilities were determined. Production of ESBL (extended spectrum β lactamase), MBL (metallo β lactamse), AmpC and KPC (Klebsiella pneumonia carbapenemase) was detected phenotypically by the use of EDTA, PBA, cloxacillin, and DDSTs. In-vitro competition assays were performed using E. coli isolates with different combinations of bacterial resistance. A total of 117 isolates was obtained with 71.8% E. coli, 7.7% of which were ESBL and 5.1% AmpC producers. Sensitive E. coli isolates out-competed all other isolates when in competition, followed sequentially by ESBL, AmpC, and OXA-48 (oxacillin) producers. This study shows an advantage of sensitive E. coli strains obtained from fecal samples to out-compete resistant strains in specific in-vitro conditions. This ability could be exploited in the elimination of MDR organisms from the gut flora, after further investigation.

Characterisation of extended-spectrum β-lactamase and AmpC β-lactamase-producing Enterobacteriaceae isolated from companion animals in New Zealand

AIMS: To assess the occurrence of, and characterise, extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC)-producing Enterobacteriaceae isolated by veterinary diagnostic laboratories from infection sites in companion animals in New Zealand.METHODS: Selected Enterobacteriaceae isolates were submitted by seven New Zealand veterinary diagnostic laboratories. They were isolated from infection sites in companion animals between June 2012 and June 2013, and were resistant to amoxicillin-clavulanic acid, fluoroquinolones, or any combination of two or more antimicrobials. Based on disk diffusion test results, the isolates were phenotypically categorised according to production of ESBL and AmpC. Genes for ESBL and AmpC production were amplified by PCR and sequenced. Escherichia coli isolates were also typed by multilocus sequence typing.RESULTS: A total of 115 isolates matching the inclusion criteria were obtained from the participating laboratories, of which 74 (64%) originated from dogs and 29 (25%) from cats. Seven bacterial species were identified, of which E. coli was the most common (87/115, 76%). Of the 115 isolates, 10 (9%) expressed the ESBL phenotype, 43 (37%) the AmpC phenotype, and seven (6%) both ESBL and AmpC phenotypes. Of the 60 ESBL and AmpC-producing isolates, 36 (60%) were E. coli. Amongst these isolates, 27/60 (45%) were classified as multidrug resistant, compared with 15/55 (27%) non-ESBL or AmpC-producing isolates (p<0.01). Ninety five isolates were resistant to amoxicillin-clavulanic acid and 58 (61%) of these were ESBL or AmpC-producing. The predominant ESBL genes were blaCTX-M-14 and blaCTX-M-15, and the dominant plasmid-encoded AmpC gene was blaCMY-2. Thirty-eight E. coli multilocus sequence types (ST) were identified, and the most prevalent were ST12 (12/89, 13%), ST131 (6/89, 7%) and ST648 (6/89, 7%). ESBL and AmpC-producing isolates accounted for 35/1,082 (3.2%) of the Enterobacteriaceae isolated by one laboratory network over the study period.CONCLUSIONS AND CLINICAL RELEVANCE: ESBL and AmpC-producing Enterobacteriaceae were associated with clinical infections in companion animals in New Zealand, and were often multidrug resistant. In this study, these organisms accounted for <5% of all Enterobacteriaceae isolated from infection sites by one laboratory network, but their prevalence among isolates resistant to amoxicillin-clavulanic acid was 61%. Therefore routine secondary testing for ESBL and AmpC production by Enterobacteriaceae that are resistant to amoxicillin-clavulanic acid in primary testing could improve the accuracy of definitive antimicrobial therapy in companion animals in New Zealand.

Antibiotic-Resistant Extended Spectrum ß-Lactamase- and Plasmid-Mediated AmpC-Producing Enterobacteriaceae Isolated from Retail Food Products and the Pearl River in Guangzhou, China.

We conducted a survey in 2015 to evaluate the presence of extended spectrum β-lactamase (ESBL)- and plasmid-mediated AmpC-producing Enterobacteriaceae in retail food and water of the Pearl River in Guangzhou, China, as well as their antibiotic resistance profiles. Samples (88 fresh food samples and 43 water samples) from eight different districts were analyzed by direct plating and after enrichment. Multidrug-resistant strains were found in 41.7 and 43.4% of food and water samples, respectively. ESBLs were found in 3.4 and 11.6% of food and water samples, respectively, and AmpC producers were found in 13.6 and 16.3% of food and water samples, respectively. Molecular characterization revealed the domination of blaCTX−Mgenes; plasmidic AmpC was of the type DHA-1 both in food and water samples. Thirteen of Fifty one β-lactamase-producing positive isolates were detected to be transconjugants, which readily received the β-lactamase genes conferring resistance to β-lactam antibiotics as well as some non-β-lactam antibiotics. These findings provide evidence that retail food and the river water may be considered as reservoirs for the dissemination of β-lactam antibiotics, and these resistance genes could readily be transmitted to humans through the food chain and water.

Panel strain of Klebsiella pneumoniae for beta-lactam antibiotic evaluation: their phenotypic and genotypic characterization.

Klebsiella pneumoniae is responsible for numerous infections caused in hospitals, leading to mortality and morbidity. It has been evolving as a multi-drug resistant pathogen, acquiring multiple resistances such as such as horizontal gene transfer, transposon-mediated insertions or change in outer membrane permeability. Therefore, constant efforts are being carried out to control the infections using various antibiotic therapies. Considering the severity of the acquired resistance, we developed a panel of strains of K. pneumoniae expressing different resistance profiles such as high-level penicillinase and AmpC production, extended spectrum beta-lactamases and carbapenemases. Bacterial strains expressing different resistance phenotypes were collected and examined for resistance genes, mutations and porin alterations contributing to the detected phenotypes. Using the Massive parallel sequencing (MPS) technology we have constructed and genotypically characterized the panel strains to elucidate the multidrug resistance. These panel strains can be used in the clinical laboratory as standard reference strains. In addition, these strains could be significant in the field of pharmaceuticals for the antibiotic drug testing to verify its efficiency on pathogens expressing various resistances.

Citrobacter as a uropathogen, its prevalence and antibiotics susceptibility pattern

Introduction: Urinary tract infection (UTI) continues to be the most common infection diagnosed in outpatients as well as in hospitalized patients. Citrobacter spp. is an emerging urinary pathogen. The present study assessed the prevalence and antibiotic sensitivity pattern of Citrobacter spp. in patients admitted to or attending outpatient departments with Complain of UTI in a tertiary care hospital. Materials and Methods: A total of 36,250 urine samples were included in the study. UTI was confirmed in 7099 samples. Of these 246 (3.46%) had UTI due to Citrobacter spp. Identification was by conventional biochemical methods. Antimicrobial susceptibility testing was performed using Kirby-Bauer disc diffusion method as recommended by Clinical and Laboratory Standards Institute M2-A9. Multidrug-resistant (MDR) was defined as resistance to more than two groups of drugs. Extended spectrum beta-lactamase (ESBL), AmpC, and metallo beta-lactamase (MBL) were detected by phenotypic methods. Results: Females predominated in the study 193 (78.4%) with 53 (21.5%) males. Most of the isolates were in the age group 21-30 and 31-40. 49 (19.9%) patients were inpatients, and 197 (80.08%) were outpatients. Aminoglycosides had a better spectrum of antimicrobial sensitivity (70.2%) with 85.2% isolates sensitive to amikacin. Fluoroquinolones had a poor activity against Citrobacter (46% sensitive). Ureidopenicillins had poor efficacy (15.4%). Out of the 246 strains, 129 (52.4%) were MDR, 61 (24.7%) of which were ESBL producers and 65 (26.4%) were AmpC producers. Most of the ESBL producing strains were isolated from inpatients. No MBL were identified in this study. Conclusion: Citrobacter though not uncommon isolate is posing a problem due to its MDR character. Infection control practices should be observed strictly, and any type of unnecessary instrumentation should be avoided.

Neonatal sepsis: Aetiological agents and risk factors

Introduction: India accounts for 30% of neonatal deaths globally. Bacterial sepsis is a major cause of morbidity and mortality in newborns. Prompt detection of microorganisms and early institution of therapy are of paramount importance. Materials and Methods: The study was conducted in the Department of Microbiology over a period of one year. Two samples of blood were collected under aseptic precautions, 1 ml each was added to conventional blood culture bottle with biphasic media and paediatric BacT/ALERT bottle. Microorganisms were identified by Gram staining, standard biochemical tests and appropriate antibiograms. The common microorganisms responsible for early- and late-onset neonatal sepsis were identified, and the resistant strains were studied in detail. The main clinical presentations and maternal and neonatal risk factors associated with neonatal sepsis were identified and statistically correlated. Results: Of the 233 newborns, 44 (18.9%) were culture positive, with higher incidence of sepsis in low birthweight male babies. Of the 44 isolates, 31 (70.5%) were Gram-negative organisms, with Klebsiella pneumoniae subspecies pneumoniae (45.5%) being the most common isolate. The prevalence of extended-spectrum beta-lactamase production in this study was 54.8%. Screening for AmpC production showed that 25.8% of the isolates were positive. The maternal risk factor of premature rupture of membrane of more than 18 h was seen in the case of 74 babies (31.8%), and 138 (59.2%) babies had prematurity as neonatal risk factor leading to sepsis. Conclusion: As the aetiological agents in neonatal sepsis vary in different circumstances and antimicrobial resistance due to different mechanisms is prevalent, antibiotic usage should be based on culture and sensitivity results.

Acquisition of Carbapenem Resistance by Plasmid-Encoded-AmpC-Expressing Escherichia coli.

Although AmpC β-lactamases can barely degrade carbapenems, if at all, they can sequester them and prevent them from reaching their targets. Thus, carbapenem resistance in Escherichia coli and other Enterobacteriaceae can result from AmpC production and simultaneous reduction of antibiotic influx into the periplasm by mutations in the porin genes. Here we investigated the route and genetic mechanisms of acquisition of carbapenem resistance in a clinical E. coli isolate carrying blaCMY-2 on a plasmid by selecting for mutants that are resistant to increasing concentrations of meropenem. In the first step, the expression of OmpC, the only porin produced in the strain under laboratory conditions, was lost, leading to reduced susceptibility to meropenem. In the second step, the expression of the CMY-2 β-lactamase was upregulated, leading to resistance to meropenem. The loss of OmpC was due to the insertion of an IS1 element into the ompC gene or to frameshift mutations and premature stop codons in this gene. The blaCMY-2 gene was found to be located on an IncIγ plasmid, and overproduction of the CMY-2 enzyme resulted from an increased plasmid copy number due to a nucleotide substitution in the inc gene. The clinical relevance of these genetic mechanisms became evident from the analysis of previously isolated carbapenem-resistant clinical isolates, which appeared to carry similar mutations.

A Study on Phenotypic Characterization of Carbapenemases among Gram Negative Bacilli in a Tertiary Care Hospital

Introduction: The emerging resistance to Carbapenems which are generally considered as life saving drugs to treat infections caused by ESBL and AmpC producing bacteria, has become a serious issue worldwide. It is therefore necessary to detect Carbapenemases to limit the spread of multidrug resistant organisms for effective Antibiotic surveillance and Infection Control in the Hospital. Aim & Objectives: To detect the presence of MBL, KPC Carbapenemase and their Co-Existence among the Carbapenem resistant clinical isolates of Gram Negative Bacilli Materials & Methods: The present study was carried out in a Tertiary care hospital; to detect Carbapenamases among Gram Negative Bacilli by Inhibitor based combined Disc tests in which Phenylboronic Acid and Dipicolinic acid are incorporated onto Meropenem discs. Results: A Total of 718 strains of Gram Negative Bacilli comprising of 516 strains of Enterobacteriaceae and 202 Non-fermenters were included in the study. Out of these 718 strains, 89 strains were resistant to carbapenems, of which 2.5% (18 /718) were KPC (Klebsiella Pneumoniae Carbapenemase –class A) producers, 8.08%(58/718) were MBL (Metallo Beta Lactamases-class B) producers .Co-existence of MBL and KPC was observed in 1.25% (9 /718) of isolates and no mechanism was detected in 4 isolates. Conclusion: Inhibitor based combined disc test is simple and cost effective phenotypic test for detecting Carbapenem resistance in the Laboratory. Antibiotic stewardship programme has to be implemented in Hospital to achieve good Infection control for better patient outcome and reduce the health care costs.

Molecular characterization and resistance profile of nosocomial Acinetobacter baumannii intensive care unit of tertiary care hospital in Bangladesh.

This study was designed to investigate the resistance profile along with the genetic background of resistance to beta-lactam antibiotics among the nosocomial A. baumannii in Bangladesh. A. baumannii was confirmed by detecting blaoXA-51-like. Antibiotic susceptibility was determined by disk diffusion method. Agar dilution method was used to determine MIC of ceftazidime and imipenem. All A. baumannii were phenotypically screened for ampC, ESBL and MBL production. Genetic markers of antibiotic resistance. such as blaampC, blaOXA-51, 23, 40, 58 and 143, blaKPc, blaMp, blavi and blaNDM-j, genetic environment around blaADc and ISAbal upstream of blaoXA, were evaluated by PCR. Twenty-four (96%) A. baumannii were considered as MDR. 96% A. baumanii were resistant to amoxiclav, ceftazidime, ciprofloxacin and cefoxitin, 92% to cefotaxime and piperacillin-tazobactam, 88% to cefepime, amikacin and imipenem, 52% to sulbactam- cefoperazone and 40% were resistant to aztreonam. Everything were sensitive to colistin. The distribution of several beta-lactamase genes such as blaoxa-51 (100%), blaADC-like (92%), blaNDM-i (92%), EBC group (84%), blaoxa-23 (76%), blavm (72%), blacpc (44%), DHA group (24%), blaoxa-58 (16%), ACC group (8%) and CIT group (4%) were observed among the 25 A. baumannii. This is the first reported plasmid mediated ampC beta-lactamases in A. baumannii. blaoxa-51 was positive in 100%, blandm-i in 95.45%, blaoxa-23 in 77.27%, blavim in 72.73%, blakpc in 50% and blaOXA-58 in 18.18% of imipenem resistant isolates. MDR profile of nosocomial A. baumannii would highlight the importance of standard guideline of antimicrobials use and infection control policy in the hospitals of Bangladesh.

Detection of AmpC β-lactamase and adherence factors in uropathogenic Escherichia coli isolated from aged patients

Escherichia coli mediated urinary tract infection has been reported to be most prevalent among patients of different class, gender and ages. Currently, multidrug resistant E. coli harboring several virulence factors are most perilous threats for patients especially for elders. The aim of this study was to determine the antibiotic resistance pattern, co-resistance and phenotypic virulence factors present in uropathogenic E. coli isolated from aged patients. Thirty-nine E. coli isolates were collected during May–June 2014 from patients between 50 to 80 years of age. Experiments have been carried out to determine the antibiotic resistance, co-resistances and phenotypic adherent factors present in each isolate. Clonal relatedness was also determined in the AmpC positive uropathogenic E. coli (UPEC). 97.43% isolates were found to be multidrug resistant and 41.02% of them were AmpC producer. AmpC producer group showed higher multiple antibiotic resistance index than AmpC non-producer (p value < 0.01) group. Interestingly, adherence factor Type 1 fimbriae were found among 84.61% of total isolates which were more prevalent in elderly female patients than males. Biofilm production studies revealed that 84.61% of total isolates are more common in elderly males. This study adds value for the proper empiric selection of antibiotic therapy as well as calls for continuous monitoring of the incidence of drug resistance virulent uropathogenic E. coli mediated urinary tract infection in elderly patients.

Prevalence of AmpC β-lactamase among Gram-negative bacteria recovered from clinical specimens in Benin City, Nigeria

Purpose: Infections caused by AmpC-positive bacteria results in high patient morbidity and mortality making their detection clinically important as they cannot be detected in routine susceptibility testing. This study aim to determine the prevalence of AmpC β-lactamase among Gram negative bacteria recovered from clinical specimens in Benin City, Nigeria.Methods: A total of 256 consecutive and non-repetitive Gram negative bacteria were recovered from various clinical specimens. The prevalence of AmpC β-lactamase was determined using a combination of disc antagonism test and cefoxitin-cloxacillin inhibition test. Disc susceptibility test was performed on all isolates using standard techniques.Results: Cefoxitin-cloxacillin inhibition test detected more AmpC β-lactamase than other tests. The prevalence of AmpC β-lactamase did not differ significantly between both genders and between inpatients and out-patients (p&gt;0.05). Isolates recovered from sputum had significantly higher prevalence of AmpC β-lactamase producers compared with isolates from other clinical specimens (p=0.0484). The prevalence of AmpC production was significantly higher among isolates of Pseudomonas aeruginosa than other isolates (p = 0.0085). Isolates that produced AmpC β-lactamase were more susceptible to the test cephalosoprins.Conclusion: An overall prevalence of AmpC β-lactamase (15.23 %) was observed in this study. Pseudomonas aeruginosa was the most prevalent producer of AmpC enzymes. Prudent use of antibiotics is advocated.Keywords: AmpC β-lactamase producers, antibiotics utilization, prevalence, Pseudomonas aeruginosa, routine susceptibility testing

Multidrug-resistant Enterobacteriaceae nosocomial uropathogens at Menoufia University Hospitals: phenotypic characterization and detection of resistance genes using real-time PCR

Objectives The aims of this study were as follows: to determine the incidence of multidrug-resistant Enterobacteriaceae isolated from the urine of patients admitted in different departments at Menoufia University Hospitals; to detect the presence of extended spectrum β-lactamases (ESβLs), AmpC β-lactamases, and carbapenemases among the isolated pathogens using phenotypic methods; and to investigate the presence of bla KPC and bla NDM genes using real-time PCR. Background Urinary tract infections are the most common type of nosocomial infections. ESβLs and AmpC β-lactamases are clinically significant because they may confer resistance to multiple classes of antibiotics. Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other β-lactams. Materials and methods This study included 260 urine samples collected from 260 patients. ESβL production was detected on the basis of resistance to third-generation cephalosporins and aztreonam and then confirmed using the combined disk test. AmpC production was detected using the cefoxitin disk test and confirmed using the AmpC disk test. Carbapenemases were detected on the basis of resistance to imipenem, meropenem, and ertapenem, and then confirmed using the modified Hodge test, the phenylboronic acid combined disk test, and the imipenem/EDTA combined disk test. Carbapenemase gene (bla KPC and bla NDM) detection was performed using real-time PCR. Results Carbapenem resistance was 42.5% to ertapenem, 40.8% to meropenem, and 45% to imipenem. Risk factors associated with Hospital aquired infection were hospital stay of 14 days or more, exposure to invasive procedures, and comorbid conditions. ESβL production occurred in 55.8% and AmpC occurred in 30.8% of Enterobacteriaceae spp. Carbapenemase production was detected in 87%, Klebsiella pneumoniae carbapenemase (KPC) was detected in 37%, metallo-β-lactamase was detected in 54%, and bla KPCgene was detected in 24% of imipenem-resistant Enterobacteriaceae spp. However, bla NDM gene was not detected in all tested isolates. Conclusion Surveys of the prevalence, antibacterial susceptibility patterns, and identification of resistance patterns of bacterial isolates are important for determining appropriate empirical therapy for infections in critically ill patients.

Phenotypic and molecular characterization of multidrug-resistant Enterobacteriaceae clinical isolates from intensive care units at Menoufia University hospitals

Objectives The objectives of the study were to determine the prevalence of multidrug-resistant Enterobacteriaceae spp. isolated from different ICUs at Menoufia University hospitals; to detect the presence of extended-spectrum β-lactamases (ESβLs), AmpC β-lactamases, and carbapenemases among the isolated pathogens by using phenotypic methods; and to investigate the presence of blaKPCand blaNDMresistance genes using real-time PCR. Background ESβL producers are associated with increased morbidity and mortality, especially among patients in ICUs. Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other β-lactams. Materials and methods This study involved 270 clinical specimens from 160 patients. ESβL production was detected by resistance to third-generation cephalosporins and aztreonam and confirmed by combined disk test. AmpC production was detected by cefoxitin disk test and confirmed by AmpC disk test. Carbapenemases were detected by resistance to imipenem, meropenem, and ertapenem, and then confirmed by modified Hodge test, phenylboronic acid combined disk (PBA-CD) test, and imipenem/EDTA combined disk (IPM/EDTA-CD) test. Carbapenemase gene (blaKPCand blaNDM) detection was performed by real-time PCR. Results In all, 52.89% of Enterobacteriaceae isolates were extreme drug resistant, 26.45% were pandrug resistant, and 14.88% were multidrug resistant. Carbapenem resistance was 60.33% for imipenem, 65.29% for meropenem, and 85.95% for ertapenem. Risk factors associated with ICUs were ICU stay greater than and equal to 14 days, exposure to invasive procedures, and comorbid conditions. ESβL production occurred in 41.32% and AmpC occurred in 32.23% of Enterobacteriaceae spp. Carbapenemase production was detected in 69.86%; Klebsiella pneumoniae carbapenemase (KPC) was detected in 60.27%; and metallo-β-lactamase was detected in 75.34%. blaKPC gene was detected in 26.03% of imipenem-resistant Enterobacteriaceae spp., whereas blaNDMgene was not detected in all tested isolates. Conclusion Surveys of the prevalence, antibacterial susceptibility patterns, and identification of resistance patterns of bacterial isolates are important for determining appropriate empirical therapy for infections in critically ill patients.

Bulgecin A as a β-lactam enhancer for carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Acinetobacter baumannii clinical isolates containing various resistance mechanisms.

Genetic screening of Pseudomonas aeruginosa (PSDA) and Acinetobacter baumannii (ACB) reveals genes that confer increased susceptibility to β-lactams when disrupted, suggesting novel drug targets. One such target is lytic transglycosylase. Bulgecin A (BlgA) is a natural product of Pseudomonas mesoacidophila and a lytic transglycosolase inhibitor that works synergistically with β-lactams targeting PBP3 for Enterobacteriaceae. BlgA also weakly inhibits di-Zn2+ metallo-β-lactamases like L1 of Stenotrophomonas maltophilia. We hypothesized that because of its unique mechanism of action, BlgA could restore susceptibility to carbapenems in carbapenem-resistant PSDA (CR-PSDA) and carbapenem-resistant ACB, as well as ACB resistant to sulbactam. A BlgA-containing extract was prepared using a previously published protocol. CR-PSDA clinical isolates demonstrating a variety of carbapenem resistance mechanisms (VIM-2 carbapenemases, efflux mechanisms, and AmpC producer expression) were characterized with agar dilution minimum inhibitory concentration (MIC) testing and polymerase chain reaction. Growth curves using these strains were prepared using meropenem, BlgA extract, and meropenem plus BlgA extract. A concentrated Blg A extract combined with low concentrations of meropenem, was able to inhibit the growth of clinical strains of CR-PSDA for strains that had meropenem MICs ≥8 mg/L by agar dilution, and a clinical strain of an OXA-24 producing ACB that had a meropenem MIC >32 mg/L and intermediate ampicillin/sulbactam susceptibility. Similar experiments were conducted on a TEM-1 producing ACB strain resistant to sulbactam. BlgA with ampicillin/sulbactam inhibited the growth of this organism. As in Enterobacteriaceae, BlgA appears to restore the efficacy of meropenem in suppressing the growth of CR-PSDA and carbapenem-resistant ACB strains with a variety of common carbapenem resistance mechanisms. BlgA extract also inhibits VIM-2 β-lactamase in vitro. BlgA may prove to be an exciting adjunctive compound to extend the life of carbapenems against these vexing pathogens.

Identification of Extended-Spectrum β-Lactamase Genes and AmpC-β-Lactamase in Clinical Isolates of Escherichia coli Recovered from Patients with Urinary Tract Infections in Kerman, Iran

Background: Escherichia coli is the main causative pathogen in urinary tract infections (UTIs). Antibiotic resistance in this bacterium is an important problem in public health. Objectives: The aim of this study was to identify the blaTEM, blaSHV, blaOXA, and blaPER genes and AmpC-β-lactamase in clinical isolates of E. coli recovered from patients with UTIs in Kerman, Iran. Methods: E. coli isolates (N = 105) were analyzed for their antibiotic susceptibility with the disk diffusion method. ESBL and AmpC-producing isolates were detected using phenotypic methods. PCR was used to identify the blaTEM, blaSHV, blaOXA and blaPER genes in ESBL and AmpC-positive isolates. Results: More than 50% of the isolates were multi-drug resistant. The prevalence of ESBLs, AmpC-β-lactamase, blaTEM and blaOXA in the inpatient isolates was 37.2%, 2%, 37.2% and 5.8%, respectively. Further, the prevalence of ESBLs, blaTEM, blaSHV and blaOXA in the outpatient isolates was 42.5%, 24%, 5.5% and 1.8%, respectively. Conclusions: The prevalence of ESBL-producing E. coli strains in the community (outpatients) is higher than that in inpatients in Kerman, Iran. An outbreak of ESBL-producing isolates in the community can be a serious problem for public health, as resistance to other classes of antibiotics such as aminoglycoside and fluoroquinolones is often related with ESBL and AmpC production, therefore, detection of ESBL and AmpC-producing isolates in the community and hospitals is very important for the treatment and prevention of such isolates.