Ceftazidime-resistant Isolates Research Articles

Roles of intrinsic and acquired resistance determinants in multidrug-resistant clinical Pseudomonas aeruginosa in Bangladesh

Introduction: Pseudomonas aeruginosa is an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.) pathogen and one of the leading etiologies in multiple nosocomial infections. Treatment of P. aeruginosa is becoming increasingly difficult due to its ever-increasing antibiotic resistance trends. This study investigated clinical multidrug resistance (MDR) P. aeruginosa (MDR-PA), their intrinsic resistance determinants, including the presence of chromosomal AmpC β-lactamase (Ampicillinase), decreased expression of outer membrane porin protein OprD and selected acquired β-lactamase resistance genes.
 Methods: Out of 238 clinical specimens, including urines from urinary tract-infected patients, wound swabs, burn swabs, and catheter aspirates, were collected from two major hospitals in Savar, Dhaka, Bangladesh. Samples were inoculated with Cetrimide agar to isolate presumptive P. aeruginosa. Bacteria were identified by cultural, biochemical characterization, 16S rDNA sequencing, and phylogenetic analysis. Virulence-associated genes of P. aeruginosa, namely, toxA, lasB, and plcH, were identified by polymerase chain reaction (PCR). Antibiotic susceptibilities of the isolates were investigated against ten antibiotics belonging to seven groups by disc-diffusion method followed by a selected minimum inhibitory concentration (MIC) assay. Phenotypic expression of Metallo-β-lactamases (MBLs) production was checked by the double disc synergistic test selectively among the imipenem-resistant isolates. Acquisition of β-lactam resistance trait was examined by PCR detection of bla-genes variants. Mutational loss of the OprD was analyzed by PCR to investigate intrinsic resistance determinants. Phenotypic overexpression of chromosomal AmpC was assayed with the identification of the AmpC gene by PCR. The expression level of OprD was assessed by real-time quantitative PCR (RT-qPCR).
 Results: Fifty-three P. aeruginosa was identified, with an overall isolation of 22.3% (53/238), where urine remains the most prevalent source. Virulence genes toxA, lasB, and plcH were identified in the isolates of 92.4%, 96.2%, and 94.3%. The highest phenotypic antimicrobial resistance was observed against ampicillin and ceftriaxone (100%), followed by cefotaxime (96%), tetracycline (89%), azithromycin (72%), imipenem (31%), ciprofloxacin (29%), levofloxacin (29%), gentamycin (27%) and ceftazidime (14%). The antibiogram pattern revealed 85% of isolates as multidrug-resistant, while 12% were considered extensively drug-resistant (XDR)-P. aeruginosa. The carriage of β-lactamase genes blaTEM, blaSHV, and blaOXA was detected in 4%, 2%, and 2% cephalosporin-resistant isolates, respectively. Double disc synergistic test revealed 87% of imipenem-resistant isolates expressing MBL-mediated resistance phenomenon. All seven ceftazidime-resistant isolates showed the presence of the AmpC gene with phenotypic overproduction of the AmpC enzyme, indicating AmpC-mediated ceftazidime resistance. Mutational loss of OprD was observed in 12% of phenotypically multidrug-resistant isolates, and RT-qPCR analysis revealed reduced expression of OprD porin protein at various levels in the outer membrane of multidrug-resistant isolates.
 Conclusions: This study depicts the high prevalence of MDR-PA in clinical specimens in Bangladesh. The identified intrinsic and acquired antimicrobial resistance determinants play synergistic roles in emerging MDR-PA.
 Bangladesh Journal of Medical Science Vol. 22 No. 03 July’23 Page : 489-507

Activity of imipenem/relebactam against Enterobacterales and Pseudomonas aeruginosa in Spain. SMART 2016-2020.

To determine susceptibility to the novel β-lactam/β-lactamase inhibitor combination imipenem/relebactam in clinical isolates recovered from intra-abdominal (IAI), urinary (UTI), respiratory (RTI) and bloodstream (BSI) infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) study in SPAIN during 2016 - 2020. Broth microdilution MICs for imipenem/relebactam and comparators were determined by a central laboratory against isolates of Enterobacterales and Pseudomonas aeruginosa. MICs were interpreted using EUCAST-2021 breakpoints. In total, 5,210 Enterobacterales and 1,418 P. aeruginosa clinical isolates were analyzed. Imipenem/relebactam inhibited 98.8% of Enterobacterales. Distinguishing by source of infection susceptibility was 99.1% in BSI, 99.2% in IAI, 97.9% in RTI, and 99.2% in UTI. Of intensive care unit isolates (ICU) 97.4% were susceptible and of non-ICU isolates 99.2% were susceptible. In Enterobacterales, activity against Class A, Class B and Class D carbapenemases was 96.2%, 15.4% and 73.2%, respectively. In P. aeruginosa, imipenem/relebactam was active in 92.2% of isolates. By source of infection it was 94.8% in BSI, 92.9% in IAI, 91.7% in RTI, and 93.1% in UTI. An 88.7% of ICU isolates and 93.6% of non-ICU isolates were susceptible to imipenem/relebactam. Imipenem/relebactam remained active against P. aeruginosa ceftazidime-resistant (76.3%), cefepime-resistant (73.6%), imipenem-resistant (71.5%) and piperacillin-resistant (78.7%) isolates. Of all multidrug-resistant or difficult-to-treat resistance P. aeruginosa isolates, 75.1% and 46.2%, respectively, were susceptible to imipenem/relebactam. Imipenem/relebactam showed high rates of susceptibility in Enterobacterales and P. aeruginosa isolates from different sources of infection as well as depending on patients' location (ICU or non-ICU scenarios).

Antimicrobial activities of ceftazidime/avibactam, ceftolozane/tazobactam, imipenem/relebactam, meropenem/vaborbactam, and comparators against Pseudomonas aeruginosa from patients with skin and soft tissue infections

BackgroundThe limited armamentarium against multidrug-resistant Gram-negative bacilli led to the development of a new generation of β-lactam/β-lactamase inhibitor combinations (BL/BLI). ObjectivesTo evaluate the in vitro activity of ceftazidime/avibactam, ceftolozane/tazobactam, meropenem/vaborbactam, and imipenem/relebactam against Pseudomonas aeruginosa isolates recovered from patients hospitalized with skin and soft tissue infections (SSTIs) in several countries around the world. MethodsA total of 360 P. aeruginosa isolates were consecutively collected from 47 medical centers located in Western Europe, Eastern Europe, the Asia-Pacific region, and Latin America. Susceptibility testing was performed by broth microdilution method at a monitoring laboratory. EUCAST breakpoints were applied. ResultsCeftazidime/avibactam (98.3% susceptible), ceftolozane/tazobactam (98.6% susceptible), and imipenem/relebactam (98.3% susceptible) were the most active compounds after colistin (100.0% susceptible) and retained activity against isolates nonsusceptible to piperacillin/tazobactam, meropenem, imipenem, and/or ceftazidime. Meropenem-vaborbactam was active against 94.2% of isolates. Ceftazidime/avibactam was the most active BL/BLI against meropenem-nonsusceptible (92.6% susceptible) and imipenem-resistant (93.8% susceptible) isolates, whereas ceftolozane/tazobactam was the most active BL/BLI against piperacillin/tazobactam-resistant (91.1% susceptible) and ceftazidime-resistant (91.7% susceptible) isolates. ConclusionsThe recently approved BL/BLIs demonstrated potent activity and broad coverage against contemporary P. aeruginosa isolates from patients with SSTIs.

Antibiotic Resistance Pattern of Pseudomonas aeruginosa Isolated from Clinical Specimens from a Tertiary Care Centre in Central Kerala with Special Reference to Carbapenemase Detection

Pseudomonas aeruginosa is inherently resistant to many drugs. It is now an emerging opportunistic pathogen of clinical relevance. The emergence of carbapenemases is another major concern. Initiation of appropriate therapy is of paramount importance thus highlighting the need of active surveillance for newer emerging resistance trends for better infection control. To study the resistance pattern of P. aeruginosa isolates obtained from lab specimens and to determine the production of ESBL and Carbapenemase among them. A hospital-based cross-sectional study was carried out in the Department of Microbiology, Government medical college Thrissur, among P. aeruginosa isolates obtained from lab specimens, from January 2018 – December 2018. 162 isolates were studied. Antimicrobial susceptibility testing was done by Kirby – Bauer disc diffusion method, extended-spectrum beta-lactamase (ESBL) production was confirmed by and phenotypic confirmatory disc diffusion test. Carbapenemase detection was done using the modified carbapenemase inactivation (mCIM) method. The obtained data was analysed. Among 162 isolates 83% were non-multidrug-resistant (MDR) strains and 17% were MDR strains. 22% of ceftazidime resistant isolates were ESBL producers. 6.2% isolates were resistant to imipenem. Among the imipenem resistant isolates, Carbapenemase production was seen in 30% isolates by mCIM test. According to our study, the most effective antibiotic against P. aeruginosa were imipenem and cefoperazone/sulbactam showing resistance in 6.3% and 6.9% isolates respectively. The diversity of antibiotic resistance mechanisms and the emergence of carbapenem resistance is a threat that limits treatment choices. This suggests the need for ongoing antimicrobial susceptibility studies in the future.

In vitro activity of ceftazidime-avibactam and comparators against Enterobacterales and Pseudomonas aeruginosa isolates from Central Europe and Israel, 2014–2017 and 2018

Between 2014 and 2017, 6,662 Enterobacterales and 1,953 P. aeruginosa isolates were collected by 19 centers in four central European countries and Israel. A further 2,585 Enterobacterales and 707 P. aeruginosa isolates were collected in 2018 by 28 centers in seven European countries and Israel as part of the Antimicrobial Testing Leadership and Surveillance (ATLAS) study. A central laboratory performed antimicrobial susceptibility testing using broth microdilution panels according to Clinical Laboratory Standards Institute (CLSI) guidelines. Susceptibility rates among Enterobacterales were highest to ceftazidime-avibactam (≥98.5%), colistin (≥97.3%), and meropenem (≥95.8%). Ceftazidime-resistant and multidrug-resistant (MDR) Enterobacterales subsets were highly susceptible to ceftazidime-avibactam (≥94.9%) and colistin (≥94.7%). Susceptibility rates to colistin among all P. aeruginosa were ≥97.4% and were ≥96.3% among ceftazidime-resistant and MDR subsets. Susceptibility rates to ceftazidime-avibactam were 91.9% (2014-2017), 86.3% (2018) and, in common with comparator agents, were lower among ceftazidime-resistant (≥51.7%) and MDR isolates (≥57.1%).

A Tale of β-Lactamases and Multidrug Resistance in Pseudomonas aeruginosa Isolated from Inpatients in a University Hospital

Introduction: In this era of modern medicine, antimicrobial resistance can be regarded as a major health calamity. The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa strains poses therapeutic challenges and lead to treatment failure in hospitalized patients. This study was conducted to determine various types of β-lactamases among MDR P. aeruginosa isolates recovered from hospitalized patients. Methods: This study was conducted at Tribhuvan University Teaching Hospital, Maharajgunj, Nepal. The clinical samples collected from inpatients were processed for detection of P. aeruginosa isolates and antibiotic susceptibility profile was determined. The MDR strains were identified and ceftazidime-resistant isolates were subjected for detection of extended-spectrum-β-lactamase (ESBL), metallo-β-lactamase (MBL), and Klebsiella pneumoniae carbapenemase (KPC). Results: A total of 161 P. aeruginosa isolates were recovered during the study period encompassing 73.3% (n=118) MDR isolates. The MDR isolates included 50.0% (n=59) from lower respiratory tract infections; and 39.8% (n=47) were from the intensive care unit patients. The MDR isolates showed a high resistance profile towards piperacillin, cephalosporins, and fluoroquinolones (>85%). Resistance to carbapenems and aminoglycosides were up to 80% and 60% respectively. Extended spectrum-β-lactamase, MBL, and KPC mediated resistance were seen in 34.7%, 43.6%, and 14.4% MDR isolates, respectively. Conclusion: Multidrug resistance as well as resistance mediated by β-lactamases production were high among P. aeruginosa isolates. Therefore, early detection of antimicrobial resistance and rational use of antibiotics play a critical role to fight against this MDR pathogen.

A Tale of β-Lactamases and Multidrug Resistance in Pseudomonas aeruginosa Isolated from Inpatients in a University Hospital

Introduction In this era of modern medicine, antimicrobial resistance can be regarded as a major health calamity. The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa strains poses therapeutic challenges and lead to treatment failure in hospitalized patients. This study was conducted to determine various types of β-lactamases among MDR P. aeruginosa isolates recovered from hospitalized patients.
 MethodsThis study was conducted at Tribhuvan University Teaching Hospital, Maharajgunj, Nepal. The clinical samples collected from inpatients were processed for detection of P. aeruginosa isolates and antibiotic susceptibility profile was determined. The MDR strains were identified and ceftazidime-resistant isolates were subjected for detection of extended-spectrum-β-lactamase (ESBL), metallo-β-lactamase (MBL), and Klebsiella pneumoniae carbapenemase (KPC).
 ResultsA total of 161 P. aeruginosa isolates were recovered during the study period encompassing 73.3% (n=118) MDR isolates. The MDR isolates included 50.0% (n=59) from lower respiratory tract infections; and 39.8% (n=47) were from the intensive care unit patients. The MDR isolates showed a high resistance profile towards piperacillin, cephalosporins, and fluoroquinolones (>85%). Resistance to carbapenems and aminoglycosides were up to 80% and 60% respectively. Extended spectrum-β-lactamase, MBL, and KPC mediated resistance were seen in 34.7%, 43.6%, and 14.4% MDR isolates, respectively.
 ConclusionMultidrug resistance as well as resistance mediated by β-lactamases production were high among P. aeruginosa isolates. Therefore, early detection of antimicrobial resistance and rational use of antibiotics play a critical role to fight against this MDR pathogen.

In vitro activity of ceftazidime-avibactam against Gram-negative isolates collected in 18 European countries, 2015–2017

BackgroundBetween 2015–2017, 21 850 Enterobacterales isolates and 6156 Pseudomonas aeruginosa (P. aeruginosa) isolates were collected by 77 centers in 18 European countries as part of the International Network for Optimal Resistance Monitoring (INFORM) study (which was included into the Antimicrobial Testing Leadership and Surveillance [ATLAS] study in 2018). MethodsA central reference laboratory performed antimicrobial susceptibility testing using broth microdilution panels according to Clinical and Laboratory Standards Institute guidelines. The presence of β-lactamases was confirmed using multiplex PCR assays. ResultsAmong Enterobacterales isolates, the highest rates of susceptibility were to ceftazidime-avibactam (99.0%; MIC90 0.5 mg/L), meropenem (96.3%), amikacin (95.2%), and imipenem (92.8%). All Enterobacterales organisms were highly susceptible to colistin (≥ 94.6%), apart from Proteus mirabilis, which is intrinsically resistant to colistin. Susceptibility rates among ceftazidime-resistant isolates were 95.7% for ceftazidime-avibactam and 87.9% for colistin, and 78.5% and 71.1%, respectively, among carbapenemase-positive isolates. Colistin was the only agent with activity against metallo-β-lactamases (100% susceptibility) among Enterobacterales and P. aeruginosa isolates. Overall susceptibility rates among P. aeruginosa were highest to colistin (99.5%) and ceftazidime-avibactam (92.3%), and were similar to ceftazidime-resistant isolates for colistin (98.9%) and reduced to 66.2% for ceftazidime-avibactam. Susceptibility rates among multidrug-resistant P. aeruginosa isolates were 98.9% to colistin and 71.7% to ceftazidime-avibactam. ConclusionsClinical isolates of Enterobacterales and P. aeruginosa collected from Europe, between 2015–2017, were highly susceptible to ceftazidime-avibactam, suggesting it is a useful alternative agent for patients whose treatment options may be limited. Persistent antimicrobial resistance requires continued surveillance and monitoring.

Phenotypic Assays for Detection of AmpC and MBL Producers among the Clinical Isolates of Multi Drug Resistant Pseudomonas aeruginosa

Objectives: In order to determine the prevalence of multi-drug resistance along with AmpC and metallo-β-lactamase producing P. aeruginosa, a six month cross-sectional study was carried out at Shahid Gangalal National Heart Center.Methods: A total of 756 clinical specimens were analyzed for bacteriological profile. The bacterial isolates were identified by cultural and biochemical techniques. Antibiotic susceptibility testing of the isolates was performed by Kirby-Bauer disc diffusion method. MDR isolates were screened and tested for MBL and AmpC production. Ceftazidime resistant isolates were tested for MBL and Cefoxitin resistant isolates for AmpC.Results: Among all the clinical samples analyzed, P. aeruginosa was detected in 75 samples (9.92%). Antibiotic susceptibility testing showed Imipenem as the most effective drug with susceptibility of 76% followed by Piperacillin-Tazobactam (74.7%) and Piperacillin (41.3%). Out of 75 P. aeruginosa isolates, 53 (70.6%) of them were found to be resistant to at least three out of four anti-pseudomonal agents, thus were considered as MDR. Out of 53 multi-drug resistant P. aeruginosa (MDRPA), all were resistant to ceftazidime whereas 85% (45/53) were resistant to cefoxitin. Out of 53 isolates, 11 (20.75%) showed positive result for MBL. Similarly, 7 out of 45 i.e. 13.2% were found to be AmpC producers.Conclusion: This study signified the high prevalence of MDRPA which is an alarming rate. Also multiple β-lactamase producing P. aeruginosa were detected which can further complicate the treatment options. Regular monitoring of antibiotic susceptibility and rational use of antibiotics would be helpful in eliminating the outbreaks of multiple β-lactamase producing MDRPA.

Unexpected mechanisms of resistance in Dutch Pseudomonas aeruginosa isolates collected during 14 years of surveillance

Pseudomonas aeruginosa is one of the most important causes of infection in intensive care units (ICUs). It is intrinsically resistant to many antimicrobials and easily acquires additional resistance genes via horizontal gene transfer of mobile genetic elements. In this study, 1528 P. aeruginosa isolates obtained from a Dutch national surveillance programme between the years 1998-2011 were analysed for the presence of extended-spectrum β-lactamase (ESBL) genes (blaCTX-M, blaSHV, blaTEM, blaBEL, blaPER, blaVEB and blaOXA-10) and metallo-β-lactamase (MBL) genes (blaIMP, blaVIM and blaNDM). Of the ceftazidime-resistant isolates, 6.2% tested phenotypically positive for ESBL. Moreover, a Verona integron-encoded MBL (VIM) gene was found in 3.1% of isolates that were phenotypically resistant to imipenem and/or meropenem. Multilocus sequence typing (MLST) of ESBL-positive isolates indicated ST1216, ST111 and ST622, with all blaVIM-positive isolates belonging to the ST111 clone. Although the prevalence of ESBL and MBL phenotypes in this Dutch national surveillance collection of >1500 ICU P. aeruginosa isolates was very low, all VIM-producing isolates belonged to the high risk-associated, international, clonal complex CC111, and most ESBL-producing isolates belonged to clonal complexes known for their successful spread, e.g. CC111 and CC235. These data indicate that high-risk clones of P. aeruginosa were present in the Netherlands between 1998-2011 and probably spread unnoticed throughout Dutch hospitals.

Activity of ceftazidime•avibactam against multidrug-resistance Enterobacteriaceae expressing combined mechanisms of resistance

IntroductionAntimicrobial resistance in Enterobacteriaceae is increasing worldwide and is making treating infections caused by multidrug-resistant Enterobacteriaceae a challenge. The use of β-lactam agents is compromised by microorganisms harboring extended-spectrum β-lactamases (ESBLs) and other mechanisms of resistance. Avibactam is a non β-lactam agent that inhibits clinically relevant β-lactamases, such as ESBL and AmpC. The ceftazidime•avibactam combination (CAZ-AVI) was recently approved for use in certain complicated infections, and may provide a therapeutic alternative for infections caused by these microorganisms. MethodsThe in vitro activity of CAZ and CAZ-AVI (AVI at a fixed concentration of 4mg/L) was tested against 250 clinical isolates of Enterobacteriaceae using broth microdilution. EUCAST breakpoint criteria were used for CAZ, and FDA criteria for CAZ-AVI. Clinical isolates included bacteria producing extended-spectrum β-lactamases (ESBLs) and acquired AmpC β-lactamases (AACBLs). Porin loss in Klebsiella pneumoniae was also evaluated. ResultsThe combination of AVI with CAZ displayed excellent activity against clinical isolates of ESBL-producing Escherichia coli and Klebsiella pneumoniae, rendering all the ceftazidime-resistant isolates susceptible to ceftazidime. CAZ-AVI retained activity against porin-deficient isolates of K. pneumoniae producing ESBLs, AACBLs, or both, although MIC values were higher compared to porin-expressing isolates. CAZ-AVI rendered all the ceftazidime-resistant AACBL-producing Enterobacteriaceae tested susceptible to ceftazidime. ConclusionCAZ-AVI showed potent in vitro activity against clinical isolates of Enterobacteriaceae producing ESBLs and/or AACBLs, including K. pneumoniae with loss of porins.

Efficacy of Cefiderocol against Carbapenem-Resistant Gram-Negative Bacilli in Immunocompetent-Rat Respiratory Tract Infection Models Recreating Human Plasma Pharmacokinetics.

ABSTRACTCefiderocol (S-649266), a novel siderophore cephalosporin, shows potent activity against carbapenem-resistant Gram-negative bacilli. In this study, we evaluated the efficacy of cefiderocol against carbapenem-resistant Gram-negative bacilli (Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae) in immunocompetent-rat respiratory tract infection models recreating plasma pharmacokinetics (PK) profiles in healthy human subjects. A total of 6 clinical isolates (1 cephalosporin-susceptible P. aeruginosa isolate, 1 multidrug-resistant P. aeruginosa isolate, 2 multidrug-resistant A. baumannii isolates, and 2 carbapenem-resistant K. pneumoniae isolates) were evaluated. Four-day treatment with a human exposure of 1 g ceftazidime every 8 h as a 0.5-h infusion showed potent efficacy only against a ceftazidime-susceptible isolate, not against five ceftazidime-resistant isolates harboring carbapenemase. With cefiderocol, a human exposure of 2 g every 8 h as a 3-h infusion for 4 days produced a >3 log10 reduction in the number of viable cells of these carbapenem-resistant isolates in the lungs. When the infusion time was 1 h, bactericidal activity was also observed against all isolates tested, although for 2 of 5 carbapenem-resistant isolates, a 3 log10 reduction was not achieved. The difference in efficacy achieved by changing the infusion period from 1 h to 3 h was considered to be due to the higher percentage of the dosing interval during which free-drug concentrations were above the MIC (%fTMIC), as observed for β-lactam antibiotics. These results suggest the potential utility of cefiderocol for the treatment of lung infections caused by carbapenem-resistant P. aeruginosa, A. baumannii, and K. pneumoniae strains.

A hospital based surveillance of metallo-beta-lactamase producing gram negative bacteria in Nepal by imipenem-EDTA disk method

BackgroundA rising threat of the rapid spread of acquired metallo-beta-lactamases (MBLs) among major Gram-negative pathogens is a matter of public health concern worldwide. Hence, for a low income nation like Nepal, surveillance data on MBL producing clinical isolates via a cost effective technique is necessary to prevent their dissemination as well as formulation and regulation of antimicrobial stewardship policy.MethodsThe prospective study was conducted at Nepal Medical College, Kathmandu from May to October, 2014 to assess the prevalence of MBL production among ceftazidime-resistant Gram-negative rods (GNRs) isolates. The samples were processed according to standard microbiological procedure following the Manual of clinical Microbiology. Isolated GNRs were subjected to susceptibility testing against the selected panel of antibiotics by Kirby- Bauer disc diffusion method and interpretation made in conformity with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Ceftazidime-resistant isolates were subjected to the detection of MBL production by imipenem—EDTA combined disc (CD) method.ResultsAmong the Gram-negative isolates, 5.80% (21/362) were found to be MBL positive with Acinetobacter spp. showing the highest prevalence i.e. 85.71% (18/21), followed by P. aeruginosa i.e. 14.29% (3/21). None of the other cefazidime resistant gram negative bacteria tested were found to be positive for MBL production with all the positive isolates determined to be Multidrug resistant (MDR) strains.ConclusionThis study demonstrated a higher rate of resistance among P. aeruginosa and Acinetobacter spp. to a wide variety of antibiotic categories with an additional burden of MBL production within them, warranting a need for strict surveillance and rapid detection of MBL production among the GNRs.

Occurrence of extended-spectrum β-lactamase, AmpC and virulence genes in Escherichia coli isolates from vegetable salads in Morocco

PurposeThe purpose of this paper is to assess the occurrence of the extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamase genes in 144 Escherichia coli isolates recovered from 160 vegetable salad samples.Design/methodology/approachAmong the 144 E. coli isolates recovered from 160 vegetable salads, 17 (12 percent) ceftazidime-resistant isolates were screened for ESBL production with the double disk-diffusion test. The ESBL-producing isolates were characterized for antimicrobial resistance, the presence of virulence genes and plasmid-mediated quinolone resistance (PMQR) determinants. The isolates were also subjected to phylogenetic group typing. The existence of plasmid AmpC genes and mutations in the regulatory region of the chromosomal AmpC gene was assessed using polymerase chain reaction (PCR) and sequencing. All β-lactamase isolates were further characterized by pulsed-field gel electrophoresis to determine the genetic relatedness.FindingsOverall, 17 (12 percent) of the 144 E. coli isolates studied were ceftazidime resistant. Among the 17 isolates, 13 (77 percent) were multidrug resistant and four (23.5 percent) were ESBL producers. The bla CTX-M14 was the only gene detected. Of the 12 AmpC-producing isolates, three (18 percent) harbored plasmid-encoded AmpC and sequencing analysis of the chromosomal AmpC genes revealed mutations in the promoter/attenuator region. PMQR determinants were detected in 9 (52 percent) isolates. A was the most prevalent phylogenetic group (56 percent), followed by groups B1 (31 percent), D (6 percent), and B2 (6 percent). PCR showed that six (50 percent) ESBL/AmpC-producing E. coli isolates carried one and/or two virulence genes. Pulsed-field gel electrophoresis showed no epidemiological relationship between these isolates.Originality/valueThis study places vegetable salads within the spectrum of ecological niches that may be vehicles for antibiotic-resistant bacteria/genes with clinical interest and these findings are worthy of attention as their spread to humans by ingestion cannot be dismissed.

Molecular epidemiology and characteristics of 16 cases with Stenotrophomonas maltophilia bacteraemia in pediatric Intensive Care Units.

Recently, Stenotrophomonas maltophilia has increasingly been reported as an important nosocomial opportunistic pathogen. Limited therapeutic options of S. maltophilia infections demand early identification and knowledge about the probable risk factors for controlling its spread. The present study aimed to investigate the risk factors and trend of antibiotic susceptibility, along with genetic analysis in bacteraemia cases at pediatric intensive care units (PICUs). A total of 16 S. maltophilia isolates were obtained, during 4 months from August to November 2015, from blood cultures of patients admitted to PICUs at Nemazee teaching hospital, Shiraz, Iran. S. maltophilia isolates were identified by conventional tests and confirmed by specific PCR primers. Minimum inhibitory concentrations (MICs) were determined by the MIC strip test as described by the Clinical and Laboratory Standards Institute's (CLSI) recommendation. The genetic relatedness among the isolates was assessed by ERIC-PCR. All isolates of S. maltophilia were susceptible to ciprofloxacin, co-trimoxazole and colistin, and only 1 (6.2%) isolate was resistant against ceftazidime. The MIC50/MIC90 of ciprofloxacin, trimethoprim/sulfamethoxazole, colistin and ceftazidime was 0.25/0.38 mg/mL, 0.125/0.19 mg/mL, 0.25/0.38 mg/mL, and 2/4 mg/mL, respectively. Genotypic analysis of ERIC-PCR results revealed two distinct types of pattern. Interestingly, the only ceftazidime resistant isolate showed different patterns with other isolates. Our findings indicated the importance of routine surveillance in infection control, since early detection of pathogens prevented the spread of nosocomial infections and granted effectiveness to care practices. Moreover, the results suggest that the routine drug of choice for S. maltophilia was mostly active against clinical isolates in our region.

Activity of ceftazidime–avibactam against multidrug-resistance Enterobacteriaceae expressing combined mechanisms of resistance

IntroductionAntimicrobial resistance in Enterobacteriaceae is increasing worldwide and is making treating infections caused by multidrug-resistant Enterobacteriaceae a challenge. The use of β-lactam agents is compromised by microorganisms harboring extended-spectrum β-lactamases (ESBLs) and other mechanisms of resistance. Avibactam is a non β-lactam agent that inhibits clinically relevant β-lactamases, such as ESBL and AmpC. The ceftazidime–avibactam combination (CAZ-AVI) was recently approved for use in certain complicated infections, and may provide a therapeutic alternative for infections caused by these microorganisms. MethodsThe in vitro activity of CAZ and CAZ-AVI (AVI at a fixed concentration of 4mg/L) was tested against 250 clinical isolates of Enterobacteriaceae using broth microdilution. EUCAST breakpoint criteria were used for CAZ, and FDA criteria for CAZ-AVI. Clinical isolates included bacteria producing extended-spectrum β-lactamases (ESBLs) and acquired AmpC β-lactamases (AACBLs). Porin loss in Klebsiella pneumoniae was also evaluated. ResultsThe combination of AVI with CAZ displayed excellent activity against clinical isolates of ESBL-producing Escherichia coli and Klebsiella pneumoniae, rendering all the ceftazidime-resistant isolates susceptible to ceftazidime. CAZ-AVI retained activity against porin-deficient isolates of K. pneumoniae producing ESBLs, AACBLs, or both, although MIC values were higher compared to porin-expressing isolates. CAZ-AVI rendered all the ceftazidime-resistant AACBL-producing Enterobacteriaceae tested susceptible to ceftazidime. ConclusionCAZ-AVI showed potent in vitro activity against clinical isolates of Enterobacteriaceae producing ESBLs and/or AACBLs, including K. pneumoniae with loss of porins.

Prevalence of ESBL phenotype, blaCTX-M-1, blaSHV and blaTEM genes among uropathogenic Escherichia coli isolates from 3 military hospitals of Tehran, Iran

Objective: To determine the extended-spectrum beta-lactamase (ESBL) production and prevalence of blaCTX-M-1, blaSHV and blaTEM genes among uropathogenic Escherichia coli (UPEC) isolates from 3 military hospitals of Tehran during 2015-2016. Methods: One-hundred and eleven isolates were adopted. The antibiotic susceptibility testing was conducted according to Clinical and Laboratory Standards Institute guidelines. The combine disk was used for phenotypic ESBL production. The ceftazidime MIC was conducted with the micro-broth dilution test. The PCR assay was used to detect the blaCTX-M-1, blaSHV and blaTEM genes. Results: In the broth microdilution method, 103 (92.7%) isolates showed minimal inhibitory concentration (MIC) ≥ 1 µg/mL, and also in the combined disk method, 89 (80.1% of all) were ESBL positive. On the other hand, among 91 ceftazidime resistant isolates, 86 (77.4% of all) were ESBL positive. The difference between the two methods for ESBL confirmation was not significant. The result of MIC was similar to the disk diffusion method in the detection of phenotypic ESBL production. Among ESBL producer isolates, the prevalence of blaCTX-M-1, blaSHV and blaTEM was 77.4% (n = 86), 47.4% (n = 53) and 2.4% (n = 2), respectively. These genes were amplified in a wide range MIC of ceftazidime. Conclusions: The prevalence of multi-drug resistant UPEC and ESBL positive isolates was high in military hospitals. The majority of UPEC isolates amplified blaCTX-M-I and blaSHV type β-lactamase genes. One-third of isolates were positive in presence of both these genes. There was no relation between ceftazidime MIC and presence of beta-lactamase genes.

Presence of blaPER-1 and blaVEB-1 beta-lactamase genes among isolates of Pseudomonas aeruginosa from South West of Iran

Pseudomonas aeruginosa isolates have acquired resistance to antibiotics such as novel beta-lactams. The aim of this study was to investigate the blaPER-1, blaVEB-1, and blaPSE-1 genes among isolates of P. aeruginosa among intensive care unit (ICU) patients. Sixty-five isolates were collected. The antibiotic susceptibility testing and combined disk tests were performed to detect the isolates producing extended spectrum beta-lactamases (ESBLs) among ceftazidime-resistant isolates. Polymerase chain reaction (PCR) amplification of blaPER-1, blaVEB-1, and blaPSE-1 genes was conducted. Ten (15.3%) isolates were ESBL-positive, of which 40% (n=4) belonged to males and 60% (n=6) were collected from females. Moreover, two and one isolates harbored blaPER-1 and blaVEB-1 genes, respectively.

Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.

Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose.

Role of the Outer Membrane and Porins in Susceptibility of β-Lactamase-Producing Enterobacteriaceae to Ceftazidime-Avibactam.

This study examined the activity of the novel antimicrobial combination ceftazidime-avibactam against Enterobacteriaceae exhibiting different outer membrane permeability profiles, specifically with or without porins and with or without expression of the main efflux pump (AcrAB-TolC). The addition of the outer membrane permeabilizer polymyxin B nonapeptide increased the antibacterial activities of avibactam alone, ceftazidime alone, and ceftazidime-avibactam against the characterized clinical isolates of Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae. This enhancement of activities was mainly due to increased passive penetration of compounds since inhibition of efflux by the addition of phenylalanine-arginine β-naphthylamide affected the MICs minimally. OmpF (OmpK35) or OmpC (OmpK36) pores were not the major route by which avibactam crossed the outer membranes of E. coli and K. pneumoniae. In contrast, Omp35 and Omp36 allowed diffusion of avibactam across the outer membrane of E. aerogenes, although other diffusion channels for avibactam were also present in that species. It was clear that outer membrane permeability and outer membrane pore-forming proteins play a key role in the activity of ceftazidime-avibactam. Nevertheless, the MICs of ceftazidime-avibactam (with 4 mg/liter avibactam) against the ceftazidime-resistant clinical isolates of the three species of Enterobacteriaceae studied were ≤ 8 mg/liter, regardless of outer membrane permeability changes resulting from an absence of defined porin proteins or upregulation of efflux.