Multidrug-resistant Enterobacteriaceae Isolates Research Articles

The effect of carbonyl cyanide m-chlorophenyl-hydrazine on antibiotic susceptibility in MDR Enterobacteriaceae isolates in Babylon, Iraq

Abstract Background: One of the efflux pump inhibitors is carbonyl cyanide 3-chlorophenylhydrazone (CCCP) that has often been found to increase the susceptibility of a number of multi-drug resistant (MDR) MDR bacteria, isolated from human clinical specimens. Objectives: To investigate the role of active efflux system to aminoglycoside and quinolones resistance in clinical isolates of Enterobacteriaceae using the efflux pump inhibitor CCCP. Materials and Methods: Enterobacteriaceae isolates were recovered from different clinical samples from hospitalized patients. The minimum inhibitory concentrations (MICs) of antibiotics (levofloxacin, ciprofloxacin, and gentamycin) were compared with and without the efflux pump inhibitor (CCCP) in order to confirm the effective role of the efflux pump in our isolates. Results: The results found that out of 280 clinical samples, only 134 (47.1%) isolates belonged to Enterobacteriaceae. Polymerase chain reaction (PCR) results showed that six (42.8%) out of 14 selected MDR isolates were positive for efflux pump gene oqxA. However, no isolates showed positive results for the efflux pump oqxB. The results of MIC for 14 Enterobacteriaceae isolates against these three antibiotics showed that all isolates had MIC ≥128 μg/mL in the absence of CCCP for levofloxacin, ciprofloxacin, and gentamycin. The results showed the MIC of levofloxacin and ciprofloxacin were reduced for isolates, and the growth of bacteria was inhibited in presence of the CCCP. However, all Enterobacteriaceae isolates showed high MIC values (≥128) even in the presence of the CCCP which indicates no effect the inhibitor in reducing the MIC of the isolates for Gentamycin. Conclusion: From this study, we can conclude that high prevalence of efflux pumps gene (oxqA) was detected among MDR and XDR Enterobacteriaceae isolates and the efflux pump inhibitor (CCCP) has a positive effect and improves the sensitivity of MDR isolates to ciprofloxacin, levofloxacin but not gentamicin.

Stability and genetic insights of the co-existence of blaCTX-M-65, blaOXA-1, and mcr-1.1 harboring conjugative IncI2 plasmid isolated from a clinical extensively-drug resistant Escherichia coli ST744 in Shanghai.

Co-existence of colistin, β-lactam and carbapenem in multidrug-resistant Enterobacteriaceae isolates poses a serious threat to public health. In this study, we investigated and characterized the co-occurrence of blaCTX-M-65, blaOXA-1, and mcr-1.1 strain isolated from a clinical extensively-drug-resistant Escherichia coli ST744 in Shanghai. Antimicrobial susceptibility test was carried out by agar dilution methods. Whole genome sequencing was conducted, and resistance genes, and sequence types of colistin in E. coli isolates were analyzed. Plasmid stability and amino acid mutations were assessed in E. coli isolates. A colistin resistant E. coli ST744, named ECPX221, was identified out of 145 fecal samples collected. The strain carries a 60,168 IncI2 plasmid with the mcr-1.1 gene. The strain also has blaCTX-M-65, blaOXA-1, dfrA14, qnrS1, cmlA5, arr2, ampC, aph(4)-Ia, sul1, and aadA5 resistance genes. The plasmid pECPX221 was capable of conjugation with an efficiency of 2.6 × 10-2. Notably, 45% of the transconjugants were determined as mcr-1.1-harboring in the colistin-free environment after 60 generation of passage. No mutations occurred in pmrB, mgrB, and phoPQ gene in the mcr-1.1-harboring transconjugants. Bioinformatic analysis indicated pECPX221 shared highly similar backbone with the previously reported mcr-1.1-harboring pAH62-1, pMFDS1339.1, pSCZE4, and p2018-10-2CC. Furthermore, sequencing and phylogenetic analyses revealed a similarity between other MCR-1-homolog proteins, indicating that ECPX221 was colistin resistant. The stable transferable mcr-1.1-harboring plasmid found in the E. coli ST744 strain indicated the high risk to disseminate the extensively-drug-resistance phenotype among Enterobacteriaceae.

Increasing trends of colistin resistance in patients at high-risk of carbapenem-resistant Enterobacteriaceae

Introduction Occurrence of colistin-resistant Enterobacteriaceae in response to the unregulated use of this antibiotic has been documented. This study reports an investigation of colistin resistance rates among carbapenem-resistant enterobacterial clinical isolates. Methods A total of 196 multidrug-resistant Enterobacteriaceae isolates (Klebsiella pneumoniae (n = 100), Escherichia coli (n = 89) and Enterobacter cloacae (n = 7) were selected from Gram-negative isolates over one year. Susceptibility to antimicrobials was determined using Vitek2. Broth microdilution method was used to detect colistin antimicrobial susceptibility. Identification of ESBL and carbapenemases were both done phenotypically and by PCR. Results All the studied isolates showed multidrug-resistant phenotypes with 51.5% resistance to carbapenems (meropenem, imipenem). Very low resistance rates towards tigecycline (n = 9) 4.6% were found. Thirty-nine isolates (19.9%) showed reduced susceptibility to colistin among the MDR isolates. Sixty-four isolates (32.7%) were ESBL producers. Hundred isolates (51%) were carbapenemase producers using Carba NP test. The PCR amplification results revealed that 40 isolates (20%) harboured NDM-1 and 40 isolates contained OXA-48-like gene. Coexistence of both (NDM-1 and OXA-48-like) was observed in nine (4.59%) isolates. A Statistically significant relationship was observed between carbapenem resistance and each of the followings; OXA-48 producers (p= .009), amikacin resistance (p = .000), gentamicin resistance (p = .032), tobramycin resistance (p = .000), and tigecycline resistance (p-value ≤ .001). A statistical significance was detected between ESBL-producing isolates and carbapenem susceptible isolates ESBL producers with p = 0.000. Conclusion An alarming sign is the increasing colistin resistance rates among carbapenem-resistant isolates. Aminoglycosides are still a therapeutic option to decrease the use of colistin and avoid further development of resistance. KEY MESSAGES High rates of colistin resistance among carbapenem-resistant Enterobacteriaceae. The choice of antibiotic is significantly associated with the clinical site of infection. Aminoglycosides are offered choices for treating multiple drug-resistant Enterobacteriaceae to preserve the colistin and carbapenems.

Phenotypic Detection of ESBL, AmpC, MBL, and Their Co-occurrence among MDR Enterobacteriaceae Isolates.

Background Emergence of extended-spectrum beta-lactamases (ESBLs), AmpC β-lactamases, and metallo-β lactamases (MBL), and their co-existence among members of Enterobacteriaceae pose newer diagnostic and therapeutic challenges. The present study examines the ESBL, AmpC, and MBL production by various phenotypic methods and their co-occurrence among the multidrug-resistant (MDR) Enterobacteriaceae clinical isolates. Materials and Methods Four hundred non-repetitive Enterobacteriaceae clinical isolates were collected from the Central Referral Hospital, Sikkim. The isolates were used for identification and their antibiotic susceptibility tests were performed according to the Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL was detected by double-disc synergy test (DDST) and phenotypic confirmatory disc-diffusion test (PCDDT), AmpC detection by AmpC E-test, and boronic acid disc diffusion (BD) test. MBL was detected using the imipenem–imipenem/EDTA disc and carba-NP tests. Results Around 76% were considered MDR. ESBL was seen in 58% and 50.4% based on DDST and phenotypic confirmation disc-diffusion test (PCDDT), respectively. AmpC was detected in 11.8% and 13.1% using a commercial E-test and boronic acid test, respectively. MBL were identified in 12.8% and 14.8% based on MBL imipenem-EDTA and carba-NP tests, respectively. Co-occurrence of ESBL and AmpC, ESBL and MBL, AmpC and MBL was seen in 5.2%, 11.5%, 1.3%, respectively, whereas a combination of these three β-lactamases was observed in only 0.3% of 304 MDR isolates. Conclusion The findings highlight a high prevalence of β-lactamases and their co-production among the Enterobacteriaceae , mainly in Klebsiella pneumoniae and Escherichia coli isolates. The study further highlights the necessity to identify the MDR β-lactamases stains for effective therapy in severe as well as mild bacterial infections, thereby enabling to reduce the risk of MDR in hospital and community settings.

Comparison of sensitivity to a commercial Origanum vulgare essential oil between extended-spectrum β-lactamases (ESBL-) and non-ESBL-producing Enterobacteriaceae isolates

This study aimed to evaluate the antibacterial effect of Origanum vulgare essential oil (OVEO) against ESBL- and non-ESBL-producing multidrug-resistant (MDR) Enterobacteriaceae isolates. OVEO composition was determined using Gas chromatograph-mass spectrometer (GC/MS). In the current study, the effect of OVEO was studied on seven MDR Enterobacteriaceae isolates. Antibacterial activity of OVEO was investigated by the disc diffusion assay and twofold serial dilution method. GC/MS analysis identified thymol (78.21%) as the single major component present in the OVEO. This EO showed an unexpectedly high antibacterial activity against all the studied MDR Enterobacteriaceae isolates, with inhibition zone diameters and minimum inhibitory concentration (MIC) values ranging from 28 ± 0.6 to 35 ± 0.6 mm and 0.31 ± 0.0 to 5 ± 0.0 μL/mL, respectively. However, ESBL-producing isolates were more susceptible to OVEO than non-ESBL producing isolates. This study compared, for the first time, the sensitivity to OVEO between ESBL and non-ESBL-producing Enterobacteriaceae isolates.

Colonization with multidrug-resistant Enterobacteriaceae among infants: an observational study in southern Sri Lanka

BackgroundThe timing of and risk factors for intestinal colonization with multidrug-resistant Enterobacteriaceae (MDRE) are still poorly understood in areas with high MDRE carriage. We determined the prevalence, timing, and risk factors associated with MDRE intestinal colonization among infants in southern Sri Lanka.MethodsWomen and their newborn children were enrolled within 48 h after delivery in southern Sri Lanka. Rectal swabs were collected from women and infants at enrollment and 4–6 weeks later. Enterobacteriaceae were isolated and identified as MDRE (positive for extended-spectrum β-lactamases or carbapenem resistant) using standard microbiologic procedures. We used exact methods (Fisher’s exact and Kruskal–Wallis tests) and multivariable logistic regression to identify sociodemographic and clinical features associated with MDRE intestinal colonization. Whole-genome sequencing was performed on selected MDRE isolates to identify phylogroups and antibiotic resistance-encoding genes were identified with NCBI’s AMRfinder tool.ResultsOverall, 199 post-partum women and 199 infants were enrolled; 148/199 (74.4%) women and 151/199 (75.9%) infants were reassessed later in the community. Twenty-four/199 (12.1%) women and 3/199 (1.5%) infants displayed intestinal colonization with MDRE at enrollment, while 26/148 (17.6%) women and 24/151 (15.9%) infants displayed intestinal colonization with MDRE at the reassessment. While there were no risk factors associated with infant colonization at enrollment, multivariable analysis indicated that risk factors for infant colonization at reassessment included mother colonized at enrollment (aOR = 3.62) or reassessment (aOR = 4.44), delivery by Cesarean section (aOR = 2.91), and low birth weight (aOR = 5.39). Of the 20 MDRE isolates from infants that were sequenced, multilocus sequence typing revealed that 6/20 (30%) were clustered on the same branch as MDRE isolates found in the respective mothers. All sequenced isolates for mothers (47) and infants (20) had at least one ESBL-producing gene. Genes encoding fosfomycin resistance were found in 33/47 (70%) of mothers’ isolates and 16/20 (80%) of infants’ isolates and genes encoding resistance to colistin were found in one (2%) mother’s isolate.ConclusionsOur results suggest that a substantial proportion of infants undergo MDRE intestinal colonization within 6 weeks of birth, potentially due to postnatal rather than intranatal transmission.

In vitro activity of tigecycline against multidrug-resistant Enterobacteriaceae isolates from skin and soft tissue infections

BackgroundTigecycline, a new agent against multidrug-resistant (MDR) bacteria, is especially licensed for use in complicated skin and soft tissue and intra-abdominal infections. We aimed to study the recent in vitro activity of tigecycline against MDR Enterobacteriaceae skin and soft tissue isolates. MethodsConsecutive isolates (56 Escherichia coli, 48 Klebsiella pneumoniae) were subjected to tigecycline susceptibility testing by Ezy MIC test and interpreted as per European Committee on Antimicrobial Susceptibility Testing. ResultsThe minimum inhibitory concentrations (MICs) of tigecycline ranged from 0.016 to 48 μg/mL, with MIC50 0.19 μg/mL and MIC90 1.0 μg/mL respectively. Seven (6.7%) isolates were resistant to tigecycline, all K. pneumoniae. ConclusionTigecycline remains a viable therapeutic option against MDR isolates, with excellent in vitro activity against E. coli and promising activity against K. pneumoniae. However, the limited availability of alternate therapeutic armamentarium necessitates its use with extreme judiciousness along with continuous monitoring for the emergence and spread of resistance.

Community acquired urinary tract infections among adults in Accra, Ghana.

Background: Urinary tract infection (UTI) is one of the most common bacterial infectious diseases encountered in clinical practice, and accounts for significant morbidity and high medical costs. To reduce its public health burden, there is the need for local research data to address aspects of prevention and management of UTI. The aim of this study was to investigate community-acquired UTI among adults in Accra, Ghana, including the risk factors, etiological agents, and antibiotic resistance.Methods: This was a cross-sectional study involving 307 patients clinically diagnosed with UTI at the Korle Bu and Mamprobi polyclinics in Accra. Urine specimens were collected from the study participants and analyzed by culture, microscopy, and dipstick. The bacterial isolates were identified using standard microbiological methods and tested against a spectrum of antibiotics by the Kirby Bauer method. Multidrug resistant Enterobacteriaceae isolates were screened for Extended Spectrum β-lactamase (ESBL) production by the double disc method, and isolates that tested positive were analyzed by Polymerase Chain Reaction for ESBL genes. Demographic information and clinical history of study participants were collected.Results: Based on the criteria for laboratory confirmed UTI, 31 (10.1%) of the 307 specimens were positive and the main risk factor of UTI among the study participants was pregnancy (P=0.02, OR=2.43). The most common uropathogen isolated was Escherichia coli (48.9%), followed by Klebseilla sp. (16.1%). Prevalence of resistance was highest for Piperacillin (87.1%) and Amoxicillin+Clavulanic Acid (87.1%) and lowest for Amikacin (12.9%). Prevalence of multidrug resistance among the uropathogens was 80.1% (25) and the most common ESBL gene detected was CTX-M-15.Conclusion: Pregnant women constitute the key risk population of UTI in Accra, while Amikacin remains a suitable drug for the treatment of febrile UTI. The high prevalence of multidrug resistance among the uropathogens highlights the need for surveillance of antimicrobial resistance among these pathogens.

Antibiotic Susceptibility Pattern of Enterobacteriaceae Isolated from Raw Meat and Ghanaian Coin Currencies at Cape Coast Metropolis, Ghana: The Public Health Implication

Introduction: The emergence and upsurge of Multiple Antibiotic Resistant (MDR) Enterobacteriaceae in the environment is a cause of concern as this can result in an outbreak and spread to healthcare settings. MDR Enterobacteriaceae have been associated with high morbidity and mortality due to delay in selecting and delivering active therapy in time. Aims & Objectives: The study was conducted to investigate the level of contamination of raw meat and Ghanaian coins in circulation at Cape Coast Metropolis. In all, 10 raw meat were sampled each from chevron and beef from Kotokuraba market, and 400 Ghanaian coin currencies retrieved from food vendors, students, transport operators and banks were used in this study. Materials & Methods: The Enterobacteriaceae species isolated were tested for their susceptibility to Ampicillin, Tetracycline (TET), Gentamicin (GEN), Cotrimoxazole (COT), Cefuroxime (CRX), Cefixime (CXM), Cefotaxime (CTX), Penicillin (PEN), Cloxacillin (CXC), Erythromycin (ERY) and Amikacin (AMK) antibiotics using Mueller-Hinton agar antibiotic diffusion technique. Of the isolated Enterobacteriaceae, 30% and 62% from chevron and beef respectively and 14.17%, 13.75% and 10.63% from food vendors, students and transport operators respectively showed resistance to some of the antibiotics tested. Results & Discussion: 8.6% of the Escherichia coli (E. coli) isolated from chevon were resistant to CRXr-CHLr-AMPr-COTr-GEMr and 15.5% of CRXi-CHLi-AMPr-TETr-COTr from beef. 40.0% of E. coli isolated from coin currencies were resistant to CRXr-CHLr-AMPr-TETr-CTXr, 50.0% of Enterobacter spp to CRXr-CHLr-AMPr-TETr-CTXr, 16.67% of Proteus spp to CRXi-CHLi-AMPr-TETr-AMKi, 40% Pseudomonas spp to CRXr-AMPr-TETr-AMKi and 100% of Enterobacter spp to CRXr-AMPr-TETi-CTXr. Conclusion: The multidrug-resistant Enterobacteriaceae isolates from circulating Ghanaian coins and raw meats in the Cape Coast metropolis is an indication of an impending danger which requires immediate attention to prevent a possible outbreak and spread from the society to the hospital setting.

Enterobacterial infection in Saudi Arabia: First record of Klebsiella pneumoniae with triple carbapenemase genes resistance.

Carbapenemase producing Enterobacteriaceae are emerging as important pathogens worldwide with serious effects on patients' outcome. The study aimed to investigate the emergence of carbapenemases associated with enterobacterial infection in Western region of Saudi Arabia. Clinical isolates from suspected patients with enterobacterial infection were investigated over a one-year period from four tertiary care hospitals of Makkah, Saudi Arabia. All isolates were identified using Vitek-2 system and then screened for potential carbapenemase production using disk diffusion test. Suspected isolates with reduced susceptibility to carbapenems were further investigated for blaNDM-1, blaKPC and blaOXA-48 resistant genes. Out of 120 confirmed Enterobacteriaceae isolates, Klebsiella pneumoniae and Escherichia coli comprised the largest proportion (35% and 34.2%, respectively) of encountered infections. Twenty-six (21.7%) isolates showed resistance to carbapenems, the majority of which (21/26) were K. pneumoniae. Remarkably, 17 isolates carried triple resistant genes KPC/NDM-1/OXA-48 while the other 4 carried double resistant genes (KPC/OXA-48) or (NDM-1/OXA-48). The current study revealed that the mentioned triple resistance genes have the higher incidence with significant association risk among males (COR 4.5; CI: 1.9-17.3; P = 0.018), non-Saudi nationalities (COR 4.9; CI: 1.5-19.3; P = 0.003), ICU-obtained specimens (COR 3.6; CI: 1.5-8.4; P = 0.002) and blood specimens (COR 2.8; CI: 1.1-6.9; P = 0.02). Multidrug-resistant Enterobacteriaceae isolates in particular K. pneumoniae co-harboring KPC, NDM-1 and OXA-48 genes are emerging in Western region, Saudi Arabia. This is the first record of triple carbapenemase genes co-producing K. pneumoniae associated with enterobacterial infection.

Assessment of the In Vivo Activity of SPR741 in Combination with Azithromycin against Multidrug-Resistant Enterobacteriaceae Isolates in the Neutropenic Murine Thigh Infection Model.

SPR741 is a novel agent with structural similarity to polymyxins that is capable of potentiating the activities of various classes of antibiotics. Previously published studies indicated that although Enterobacteriaceae isolates had minimal susceptibilities to azithromycin (AZM), the in vitro antimicrobial activity of AZM against Enterobacteriaceae was enhanced when it was combined with SPR741. The current study evaluated the in vivo activity of human-simulated regimens (HSR) of AZM equivalent to clinical doses of 500 mg given intravenously (i.v.) every 24 h (q24h) and SPR741 equivalent to clinical doses of 400 mg q8h i.v. (1-h infusion), alone and in combination, against multidrug-resistant (MDR) Enterobacteriaceae We studied 30 MDR Enterobacteriaceae isolates expressing a wide spectrum of β-lactamases (ESBL, NDM, VIM, and KPC), including a subset of isolates positive for genes conferring macrolide resistance (mphA, mphE, ermB, and msr). In vivo activity was assessed as the change in log10 CFU per thigh at 24 h compared with 0 h. Treatment with AZM alone was associated with net growth of 2.60 ± 0.83 log10 CFU/thigh. Among isolates with AZM MICs of ≤16 mg/liter, treatment with AZM-SPR741was associated with an average reduction in bacterial burden of -0.53 ± 0.82 log10 CFU/thigh, and stasis to 1-log kill was observed in 9/11 isolates (81.8%). Combination therapy with an AZM-SPR741 HSR showed promising in vivo activity against MDR Enterobacteriaceae isolates with AZM MICs of ≤16 mg/liter, including those producing a variety of β-lactamases. These data support a potential role for AZM-SPR741 in the treatment of infections due to MDR Enterobacteriaceae.

Antibacterial activity of Thymus vulgaris essential oil alone and in combination with cefotaxime against blaESBL producing multidrug resistant Enterobacteriaceae isolates

The aim was to evaluate the susceptibility of blaESBL producing Enterobacteriaceae to Slovakian Thymus vulgaris essential oil (TVEO) alone and in combination with cefotaxime (CTX). TVEO composition was determined by gas chromatograph-mass spectrometer (GC/MS). Susceptibility to 21 antibiotics was determined by disc diffusion assay. Genes characterization for resistance to β-lactams was accomplished by polymerase chain reaction (PCR). The antibacterial activity was investigated by standard methods. The synergistic interaction was determined by checkerboard test. Thymol (34.5%), p-cymene (22.27%) and linalool (5.35%) were the major components present in the TVEO. The identified strains were multi-drug resistant (MDR). TVEO showed high activity against all MDR strains, including blaESBL producing isolates, with inhibition zones and MIC values in the range of 24–40 mm/10μL and 2.87–11.5 μg/mL, respectively. TVEO in combination with CTX showed a synergistic action against blaSHV-12 producing Escherichia coli (FICI 0.28) and an additive effect vs ESBL producing Enterobacter cloacae (FICI 0.987).

Molecular analysis of integron gene cassette arrays associated multi-drug resistant Enterobacteriaceae isolates from poultry

The study investigated 110 Enterobacteriaceae isolates from broiler chickens isolated from Sharkia poultry farms and analyzed the isolates antimicrobial resistance and the presence of integrons as a potential basis for this resistance. Antibiotic susceptibilities against 12 different antibiotics were determined by the disk diffusion method. Prevalences and classes of integrons were then detected in multi-drug resistant (MDR) strains using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by sequencing of the variable parts. Fifty-three isolates were MDR (resistant to three or more antimicrobial agents). High resistance was detected for rifamycin (82.7%), erythromycin (67.2%), and amoxicillin-clavulanic acid (63%). Classes 1 and 2 integrons were detected in 38 of 53 MDR Enterobacteriaceae isolates of which the most common were Salmonella species (n=19), followed by Escherichia coli (12), Klebsiella pneumoniae (3), Proteus species (3), and Citrobacter freundii (1). Three isolates only harbored class 1 integrons while the remaining 35 isolates carried class 2. All class 1 integron positive isolates exhibited the same gene cassettes arrangements: 1.) dfrA12-orfF-aadA27 (1.6 kbp); 2.) aadA23 (1.0 kbp); and 3.) dfrA15 (0.8 kbp). Moreover, four different gene cassettes were identified within class 2 integrons: 1.) dfrA1-sat2-aadA30 (2 kbp) in all isolates; 2.) sat2-aadA1 (1.7 kbp) in only one isolate; 3.) catB2 (0.9 kbp) in four isolates; and 4.) a new variant of sat2 (0.65 kbp) in three isolates. Efforts should be made to introduce surveillance programs for monitoring antimicrobial resistance that could potentially be transmitted from broiler chickens to human via integrons.

Antibiotic susceptibility pattern of multidrug-resistant Enterobacteriaceae in urinary isolates and detection of suspected carbapenemase production

Context: Urinary tract infection (UTI) is one of the most common bacterial infections. Multidrug-resistant (MDR) Enterobacteriaceae producing carbapenemases pose a challenge for treatment. The modified Hodge test (MHT) detects carbapenemase production in isolates of Enterobacteriaceae. It is simple, accessible, inexpensive, and recommended by Clinical and Laboratory Standards Institute (CLSI). Aims: (1) To study the antibiotic susceptibility pattern of MDR urinary isolates of Enterobacteriaceae. (2) To determine percentage of carbapenem resistance due to carbapenemase production by using MHT. Settings and Design: Prospective laboratory-based observational cross-sectional study in a tertiary care hospital. Patients and Methods: Urine samples (clean and catheter catch) from both inpatients and outpatients were inoculated on cystine lactose electrolyte deficient agar by semi-quantitative method. Significance of growth was established; identification and susceptibility testing was done by using VITEK 2 compact (bioMerieux). Results were interpreted according to CLSI guidelines. MDR Enterobacteriaceae isolates were identified and further tested by MHT as described by CLSI. Statistical Analysis Used: Data were analyzed using Statistical Package for the Social Sciences version 20 (IBM Inc., SPSS Inc., Chicago, IL, USA) software. Descriptive statistics and Chi-square test were used. P values < 0.05 were considered statistically significant. Results: MDR was noted in 25.79% of Enterobacteriaceae isolates. Escherichia coli and Klebsiella pneumoniae together accounted for 87.3% of MDR isolates. Susceptible antibiotics were colistin (91.02%) and amikacin (51.15%). Pandrug resistance (PDR) was noted in 20 isolates. MHT positivity was noted in 72.3% of isolates. Conclusions: Majority of isolates were MHT positive indicating high carbapenemase production. Low susceptibility profile and occurrence of PDR in this setup deter the use of common empiric treatment for all suspected UTI cases.

High Prevalence of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae in Wild Fish from the Mediterranean Sea in Algeria.

We investigated the prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae among wild fish from the coast of Bejaia (Algeria) in the Mediterranean Sea. From March 2012 to August 2013, gut and gill samples of wild fish were screened for the presence of ESBL-producing Enterobacteriaceae. Strains were characterized with regard to antibiotic resistance, β-lactamase content, plasmid-mediated quinolone resistance, aminoglycoside resistance genes, and clonality (repetitive sequence-based polymerase chain reaction profiles and multilocus sequence typing). Virulence traits were performed for Escherichia coli and Klebsiella pneumoniae isolates. Of the 300 fish studied, 64 (21.3%) isolates were screened as positive for ESBL producing by the double-disc method. The isolates corresponded to E. coli, K. pneumoniae, Enterobacter cloacae, Morganella morganii, Citrobacter freundii, and Proteus vulgaris. A predominance of blaCTX-M gene was observed with a prevalence of 60.5% (n = 46). Furthermore, our study describes the association of important coresistance and virulence factors in E. coli and K. pneumoniae. Twelve of the ESBL producers carried genes of the qnr family and oqxAB gene and six carried the aac(6')-Ib-cr gene. Our results highlight for the first time the diffusion of multidrug-resistant Enterobacteriaceae isolates carrying resistance and virulence genes in fish from the Mediterranean Sea in Algeria.

High frequency of the combined presence of QRDR mutations and PMQR determinants in multidrug-resistant Klebsiella pneumoniae and Escherichia coli isolates from nosocomial and community-acquired infections.

Plasmid-mediated quinolone resistance (PMQR) determinants combined with mutations in quinolone resistance-determining regions (QRDRs) and clonal dissemination were investigated in 40 fluoroquinolone-resistant Klebsiella pneumoniae and Escherichia coli isolates from nosocomial and community-acquired infections. We observed nucleotide substitutions in gyrA (Ser83Ile, Val37Leu, Lys154Arg, Ser171Ala, Ser19Asn, Ile198Val, Ser83Tyr, Ser83Leu, Asp87Asn and Asp87Gly) and parC genes (Ser80Ile, Glu84Lys, Ala129Ser, Val141Ala and Glu84Gly). Two novel substitutions were detected in the gyrA gene (Val37Leu and Ile198Val). The presence of PMQR genes predominated in community isolates (55.5 %). In addition to the frequent presence of the class 1 integron in isolates from community-acquired infections, the genetic similarity results obtained by PFGE showed high genomic diversity. This study suggests that management of multidrug-resistant Enterobacteriaceae isolates from the community are a possible source of genetic mobile elements that carry genes that confer resistance to fluoroquinolones. More attention should be paid to the surveillance of community-acquired infections.

Fosfomycin: In vitro efficacy against multidrug-resistant isolates beyond urinary isolates.

Fosfomycin (FOF) is a bactericidal antimicrobial agent active against a range of Gram-negative bacteria, including multidrug-resistant (MDR) and metallo-β-lactamase (MBL)-producing Enterobacteriaceae. However, data are scarce regarding use of the drug beyond urinary tract infections (UTIs). In this study, susceptibility rates to FOF among 290 MDR Enterobacteriaceae isolates were analysed by gradient and disk diffusion tests and the results were compared with agar dilution according to the Clinical and Laboratory Standards Institute (CLSI). Minimum inhibitory concentrations (MICs) of imipenem (IPM) for isolates IPM-resistant/intermediate-susceptible isolates were determined by gradient test. In addition, the gradient test was used to determine MBL production. Of the 290 extended-spectrum β-lactamase (ESBL)-positive isolates, 60 (20.7%) were resistant to FOF, with rates of 9.5% for Escherichia coli, 28.0% for Enterobacter spp., 35.7% for Klebsiella spp. and 50.0% for Morganella spp. Among the 290 ESBL-positive isolates, 19 (6.6%) were resistant/intermediate-susceptible to IPM. In addition, 72.2% of extensively drug-resistant (XDR) and 61.1% of carbapenem-resistant isolates were resistant to FOF. In vitro FOF activity was higher among blood (86.9%) and genitourinary (91.7%) isolates. FOF showed excellent activity for a wide range of infections; however, further trials are necessary to evaluate its clinical efficacy. FOF presented good activity even against carbapenem-resistant isolates and may be a treatment alternative for non-UTI isolates, but should be used with caution for infections related to ESBL-producing Klebsiella spp.

High prevalence of VIM-4 and NDM-1 metallo-β-lactamase among carbapenem-resistant Enterobacteriaceae

The purpose of this study was to identify the mechanisms leading to carbapenem resistance among multidrug-resistant Enterobacteriaceae isolates recovered from hospitalized patients with nosocomial infections in Mubarak Al Kabeer Hospital, Kuwait. Fourteen carbapenem-resistant Enterobacteriaceae isolates were obtained from inpatients in different wards and intensive care units between April 2009 and February 2011. Antibiotic susceptibilities were determined using the E-test method. Genes encoding β-lactamases were characterized by specific PCR amplification, sequencing and conjugation assays. All isolates were identified as metallo-β-lactamase (MBL) producers using phenotypic and molecular methods. Eleven of the 14 isolates produced VIM-4 (six Klebsiella pneumoniae, three Escherichia coli, one Enterobacter cloacae and one Klebsiella oxytoca). Three K. pneumoniae isolates produced the MBL NDM-1 and co-produced the plasmid-encoded AmpC CMY-4. The VIM-4-producing isolates co-produced extended-spectrum β-lactamases including CTX-M-15 and some SHV derivatives. The VIM-4 gene was not transferable by conjugation studies of six selected strains. We demonstrated here the emergence of VIM-4- and NDM-1-producing isolates in the largest teaching hospital in Kuwait.

Molecular characterization of multidrug-resistant extended-spectrum β-lactamase-producing Enterobacteriaceae isolated in Antananarivo, Madagascar

BackgroundWe investigated the molecular characteristics of multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolated in community settings and in hospitals in Antananarivo, Madagascar.ResultsForty-nine E. coli, K. pneumoniae, K. oxytoca and E. cloacae ESBL-producing isolates were studied. In antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. Gene amplification analysis and sequencing revealed that 75.5% (n=37) of the isolates harbored blaCTX-M-15 and 38.7% (n=19) harbored blaSHV-12. The non-ESBLs resistance genes detected were blaTEM-1, blaOXA-1, aac(6′)-Ib,aac(6′)-Ib-cr, tetA, sul-1, sul-2, qnrA, qnrB and catB-3. We found dfrA and aadA gene cassettes in the class 1 integron variable regions of the isolates, and the combination of dfrA17-aadA5 to be the most prevalent. All blaCTX-M-15 positive isolates also contained the ISEcp1 insertion element. Conjugation and transformation experiments indicated that 70.3% of the antibiotic resistance genes resided on plasmids. Through a PCR based replicon typing method, plasmids carrying the blaSHV-12 or blaCTX-M-15 genes were assigned to either the IncFII replicon type or, rarely, to the HI2 replicon type. All isolates were subtyped by the rep-PCR and ERIC-PCR methods.Phylogenetic grouping and virulence genotyping of the E. coli isolates revealed that most of them belonged to group A1. One isolate assigned to group B2 harbored blaCTX-M-15 and five virulence genes (traT, fyuA, iutA, iha and sfa) and was related to the O25b-ST131 clone.ConclusionsOur results highlight the dissemination of multidrug resistant Enterobacteriaceae isolates in Antananarivo. These findings underline the need for a rational use of antibiotic and for appropriate methods of screening ESBL in routine laboratories in Antananarivo.

Emergence of extended-spectrum β-lactamase and fluoroquinolone resistance genes among Irish multidrug-resistant isolates

This study sought to identify mechanisms behind resistance to third-generation cephalosporins and ciprofloxacin in Irish multidrug-resistant Enterobacteriaceae isolates. The most prevalent extended-spectrum β-lactamase genes identified were bla SHV-12 and bla CTX-M-15. These were associated with the fluoroquinolone resistance genes aac(6′)-IB-cr, qnrA, and qnrB, not previously reported in Irish isolates.