Detection Of Β-lactamases Research Articles

Automated antimicrobial susceptibility testing and antimicrobial resistance genotyping using Illumina and Oxford Nanopore Technologies sequencing data among Enterobacteriaceae.

Whole-genome sequencing (WGS) enables the molecular characterization of bacterial pathogens. We compared the accuracy of the Illumina and Oxford Nanopore Technologies (ONT) sequencing platforms for the determination of AMR classes and antimicrobial susceptibility testing (AST) among 181 clinical Enterobacteriaceae isolates. Sequencing reads for each isolate were uploaded to AREScloud (Ares Genetics) to determine the presence of AMR markers and the predicted WGS-AST profile. The profiles of both sequencing platforms were compared to broth microdilution (BMD) AST. Isolates were delineated by resistance to third-generation cephalosporins and carbapenems as well as the presence of AMR markers to determine clinically relevant AMR classes. The overall categorical agreement (CA) was 90% (Illumina) and 88% (ONT) across all antimicrobials, 96% for the prediction of resistance to third-generation cephalosporins for both platforms, and 94% (Illumina) and 91% (ONT) for the prediction of resistance to carbapenems. Carbapenem resistance was overestimated on ONT with a major error of 16%. Sensitivity for the detection of carbapenemases, extended-spectrum β-lactamases, and plasmid-mediated ampC genes was 98, 95, and 70% by ONT compared to the Illumina dataset as the reference. Our results highlight the potential of the ONT platform’s use in clinical microbiology laboratories. When combined with robust bioinformatics methods, WGS-AST predictions may be a future approach to guide effective antimicrobial decision-making.

Prevalence of Waterborne blaNDM-1 Gene Producing Carbapenem-resistant Klebsiella pneumoniae from Al-Hillah River Water, Babylon Province, Iraq

The current work suggested the occurrence of blaNDM-1 gene among Klebsiella pneumoniae recovered from surface waters of the Al-Hillah River. Between January and April 2015, water samples (101) were taken from seven different area of the Al-Hillah River, Babylon province, Iraq. K.pneumoniae was reported in percentage of 35 (34.6%). The antibiotics susceptibility profile of K.pneumoniae was determined with disk diffusion assay. The most common resistance was detected for penicillins agents (ampicillin and cloxacillin) with 20(57.14%) and 17(48.57%) resistance rate, respectively. Two isolates of K.pneumoniae were carbapenem-resistant. Phenotypic screening of metallo β-lactamase detection was carried out using imipenem–EDTA double disk synergy test for carbapenem resistant isolates, 2(100%) isolates with positive result. Conventional Polymerase Chain Reaction (PCR) test was used for detection NDM-1 beta-lactamase, 1 (50%) K.pneumoniae isolate harboring this gene.

Antibacterial activity of Chinese propolis and its synergy with β-lactams against methicillin-resistant Staphylococcus aureus.

Propolis has exhibited effective antibacterial activities in preventing the growth of multiple pathogenic bacteria. However, the antibacterial activity of Chinese propolis against methicillin-resistant Staphylococcus aureus (MRSA) is almost unknown. The present study aimed to explore the antibacterial activity and action mechanism of Chinese propolis ethanol extract (CPEE) against MRSA. Thirteen compounds of CPEE were identified using HPLC-DAD/Q-TOF-MS, and none of them showed better anti-MRSA activity than CPEE. The diameter of inhibition zone (DIZ) of CPEE was 20.1mm. The minimal inhibitory concentration (MIC) of CPEE was 32mg/L, while the minimal bactericidal concentration (MBC) against MRSA was 64mg/L. Moreover, CPEE showed significant synergistic effects with β-lactam antibiotics (ampicillin and oxacillin). Nucleic acid and protein leakage assays showed that CPEE can stimulate the release of intracellular macromolecules by damaging the cell membrane integrity of MRSA. Live/dead-staining and SDS-PAGE assays further confirmed that CPEE could inhibit bacterial activities by disrupting the membrane. The reduction in PBP2a expression and β-lactamase activity, as shown by western blot and β-lactamase detection assays, suggested that CPEE was able to reverse the drug resistance of MRSA. These results demonstrated the anti-MRSA activity of CPEE was mainly due to changing the cell membrane and reversing resistance, which indicates that CPEE could be an attractive candidate for use in future food and medical applications.

Evaluation of a diagnostic algorithm for rapid identification of Gram-negative species and detection of extended-spectrum β-lactamase and carbapenemase directly from blood cultures.

To evaluate a rapid diagnostic algorithm based on MALDI-TOF MS, lateral flow immunoassays (LFIAs) and molecular testing performed directly from positive blood cultures (BCs) for Gram-negative species identification and detection of CTX-M extended-spectrum β-lactamases and main carbapenemases. Non-duplicate BCs positive to Gram-negative bacteria at microscope examination were subjected to species identification by direct MALDI-TOF MS following recovery of bacterial pellet by Rapid MBT Sepsityper® kit. Subsequently, NG-Test® CARBA 5 and NG-Test® CTX-M MULTI LFIAs were performed according to identified microbial species. Eazyplex® SuperBug CRE molecular assay was performed in cases of NG-Test® CARBA 5 negative results in patients with documented carbapenemase-producers carriage. Results of rapid diagnostic workflow were compared with those obtained by conventional diagnostic routine. Overall, the direct MALDI-TOF MS protocol allowed reliable identification to the species level of 92.1% of the 2133 monomicrobial BCs. Rate of matched identification was significantly higher for Enterobacterales (97.3%) in comparison to non-fermenting Gram-negative species (80.2%), obligate anaerobic bacteria (42.1%) and fastidious Gram-negative species (41.5%). The overall sensitivity of NG-Test® CARBA 5 and NG-Test® CTX-M MULTI was 92.2% and 91.6%, respectively. Integration of Easyplex® SuperBug CRE allowed the detection of blaKPC mutants associated with ceftazidime/avibactam resistance, reaching 100% sensitivity in carbapenemase detection. Both LFIAs and molecular testing showed no false-positive results. Algorithms based on MALDI-TOF MS, LFIAs and molecular testing may represent a cost-effective tool to timely identify Gram-negative species and detect resistance markers directly from BCs. According to local epidemiology, these results may allow antimicrobial stewardship interventions including prompt use of new approved drugs.

Role of Multidrug-resistant Pathogens in Ventilator-Associated Pneumonia in a Tertiary Care Hospital in India

Aim: Ventilator-associated pneumonia (VAP) is the second most common infection acquired in the intensive care unit (ICU). Bacteriological profiles cause VAP and their susceptibility patterns vary in different institutions. Methods: A prospective study was conducted from June 2017 to May 2018 in a tertiary care hospital as per the recent NHSN guidelines in finding the incidence of VAP and further determining the etiological agents by both conventional and automated methods. The combination disk method (Phenotypic confirmatory test), ampicillin C (AmpC) disk test, modified carbapenem inactivation method, imipenem/ethylenediamine tetraacetic acid combined disc test, and cefoxitin disk test were performed for the detection of extended-spectrum beta-lactamases (ESBL), AmpC β-lactamases, carbapenemases, metallo-beta-lactamases (MBL), and methicillin-resistant Staphylococcus aureus, respectively. Results: Among 104 patients, 31 cases developed PVAP (possible VAP) during their ICU stay; of these cases, two patients had two episodes of VAP each, and the incidence of VAP was 32%. The most common isolate was Acinetobacter baumannii (38%), followed by Pseudomonas aeruginosa (22%), Klebsiella pneumoniae (16%), and Escherichia coli (13.51%). Twenty (54%) of the 37 VAP pathogens were multidrug resistant. ESBL was produced by 40% and 67% of E. coli and K. pneumoniae, respectively. MBL was produced by 25% of P. aeruginosa. In addition, AmpC beta-lactamases were produced by 18% each of the Enterobacteriaceae and non-fermenters, respectively. One of the two S. aureus isolates was methicillin-resistant. Conclusion: The majority of VAP cases in our setting were caused by highly resistant strains. The frequency of specific multidrug resistance pathogens causing VAP may vary due to hospital, patient population, exposure to antibiotics, type of ICU patients, and changes over time, emphasizing the need for timely local surveillance data.

Direct Identification, Antimicrobial Susceptibility Testing, and Extended-Spectrum β-Lactamase and Carbapenemase Detection in Gram-Negative Bacteria Isolated from Blood Cultures.

PurposeTo shorten the turnaround time for blood culture (BC) analyses, a rapid method was developed for the direct identification, antimicrobial susceptibility testing (AST), and multidrug resistance testing of bacteria-positive BCs.Materials and MethodsThe mixtures in BC bottles were treated with the multistep centrifugation method developed here and the conventional culture-based method. The bacterial sediment obtained after centrifugation was analyzed directly with MALDI-TOF MS and Vitek 2 Compact, and AST was performed directly with the Kirby–Bauer (K–B) disk diffusion, VITEK 2 Compact, and E-test methods. Extended spectrum lactamases (ESBLs) were detected with discs containing cefotaxime, cefotaxime/clavulanate, ceftazidime, and ceftazidime/clavulanate, and carbapenemase was detected with the modified carbapenem inactivation method (mCIM) and EDTA-mCIM (eCIM).ResultsAll the results of direct testing were compared to those of the conventional methods, to evaluate the accuracy of the direct methods. The accuracies of the direct Vitek 2 Compact and MALDI-TOF MS methods were 95.5% (214/224) and 90.2% (202/224), respectively. Direct AST with K–B, Vitek 2, and E-test showed category agreement of 96.0% (2611/2721), 96.1% (2614/2721), and 97.4% (2650/2721), respectively, and the major errors and very major errors were < 2% for all three methods. In the direct determination of ESBLs, the results for cefotaxime combined with cefotaxime/clavulanate were completely consistent with those after the standard isolation method. The carbapenemase detection rate with direct mCIM and eCIM was exactly the same as that with the standard method.ConclusionThese direct procedures based on multistep centrifugation are not only highly accurate but are appropriate for clinical laboratory use because the turnaround time is shorter.

The Diagnostic Capacity of Three Phenotypic Techniques of Extended-Spectrum β-Lactamase Detection

Background: Early detection of extended-spectrum β-lactamases (ESBLs) producing bacteria is critical for infection prevention and control. Numerous phenotypic approaches and automated systems have been developed for detecting ESBL bacteria. However, there is a scarcity of data in Ethiopia regarding the most reliable, simple, and cost-effective methods for detecting ESBL-producing bacteria. This study, therefore, aimed to evaluate the diagnostic performance of three phenotypic approaches for detecting ESBL-producing bacteria. Methods: In this study, 117 isolates of Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, and Proteus mirabilis were examined. Cefotaxime (30 µg) and ceftazidime (30 µg) were used for screening ESBL enzymes. A screening breakpoints of≤27 mm and≤22 mm were used for cefotaxime (30 µg) and ceftazidime (30 µg), respectively, as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. All 117 strains were further confirmed by the Vitek 2 compact, double disk synergy, ESBL Epsilometer test, and combined disk method. The combined disk method was adopted as the reference method. Results: Out of 117 isolates, 90 (86%) had zone diameters of≤27 mm and≤22 mm for cefotaxime (30 µg) and ceftazidime (30 µg), respectively. The reference method detected 76 (65%) ESBL isolates out of 117 ones. From among the three techniques (i.e., double disk synergy, Vitek 2 compact, and ESBL Epsilometer test), the double disk synergy method demonstrated overall sensitivity and specificity of 97.4% and 97.6%, respectively. Vitek-2, cefotaxime, and ceftazidime Epsilometer test indicated indeterminate results of 6.8%, 6.8%, and 5.1% respectively. Conclusion: Double disk synergy was found to have the highest sensitivity and specificity for detecting ESBL isolates with no indeterminate results.

Detection and Molecular Characterization of Extended-spectrum β-lactamase Producing E. coli and Klebsiella spp. Isolates of Cattle Origin in Eastern Plain Zone of Uttar Pradesh

Background: Antibiotics are widely used in animals and human for treatment without referring an antibiogram that resulted into the proliferation of ESBL producing strains. Nowadays resistance to β-lactam groups of antibiotic is expanding rapidly worldwide and threatening the public healthcare due to limited treatment options. Therefore this study aimed to detect ESBL resistance genes in E. coli and Klebsiella spp. isolated from various sources of cattle in this area of study. Methods: Total 240 samples were collected during August, 2019 to June, 2020, from two districts of Eastern plain zone of Uttar Pradesh. E. coli and Klebsiella spp. isolates were confirmed using uidA and 16S rRNA gene respectively. In vitro antibiotic sensitivity test was performed using disc diffusion method. ESBL producing isolates was confirmed by DDST, ESBL E-strip and PCR analysis by targeting (bla-CTX-M-1, bla-CTX-M-9, bla-TEM and bla-SHV) genes. Result: PCR analysis of these isolates confirmed 135(56.25%) as E. coli and 16(6.67%) as Klebsiella spp. Antibiotics found to be most resistant were ampicillin (89.40%) followed by cefotaxime (72.18%), cefpodoxime (71.52%), ceftriaxone (66.9%), aztreonam (61.0%) and ceftazidime (54.30%). Total 101(66.88%) isolates were confirmed as ESBL producers using DDST and 92(60.92%) by ESBL E-strip test. ESBL genes were detected in 87(57.61%) isolates by PCR analysis and among them, bla-CTX-M-1 was found most dominant gene.

Molecular Detection of Integrons, Colistin and β-lactamase Resistant Genes in Salmonella enterica Serovars Enteritidis and Typhimurium Isolated from Chickens and Rats Inhabiting Poultry Farms.

The rapid growth of multidrug-resistant Salmonella is a global public health concern. The aim of this study was to detect integrons, colistin and β-lactamase resistance genes in Salmonella enteritidis and typhimurium. A total of 63 isolates of S. enteritidis (n = 18) and S. typhimurium (n = 45) from fecal samples of layers and rats at chicken farms were screened for antibiotic resistant genes. Conventional PCR was performed for the detection of integrons (classes 1, 2, and 3), colistin (mcr-1-5) and β-lactamase (blaCTX-M, blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaCTX-M-15, blaTEM, blaSHV, and blaOXA) resistant genes. Of these isolates, 77% and 27% of S. typhimurium and S. enteritidis harboured the mcr-4 encoded gene for colistin, respectively. The prevalence of class 1 integrons for S. typhimurium and S. enteritidis was 100% for each serovar, while for class 2 integrons of S. typhimurium and S. enteritidis it was 49% and 33% respectively, while class 3 integron genes was not detected. Our study also detected high levels of β-lactamase encoding genes (bla gene), namely blaCTX-M, blaCTX-M-1, blaCTX-M-9 and blaTEM from both S. typhimurium and S. enteritidis. This, to our knowledge, is the first report of mcr-4 resistance gene detection in Salmonella serovars in South Africa. This study also highlights the importance of controlling rats at poultry farms in order to reduce the risk of transmission of antibiotic resistance to chickens and eventually to humans.

Novel approach for rapid detection of extended spectrum β-lactamase and metalloid-β-lactamase using drug susceptibility testing microfluidic device (DSTM)

Background/purpose: Rapid detection of β-lactamases is important in a recent situation where resistant bacteria are increasing. By using the drug susceptibility testing microfluidic device (DSTM), rapid screening of extended spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) has become possible. Methodsβ-lactams and β-lactamase inhibitors were pre-fixed in the DSTM for use. A bacterial suspension in Mueller-Hinton broth (McF 0.25) was introduced into the device, and the effects of β-lactamase inhibitor on morphological changes caused by β-lactam were evaluated after 3 h incubation. ResultsClinical isolates genetically confirmed to produce β-lactamase were used. Of the 84 ESBL-producing strains, 80 strains (95%) turned to be ESBL positive, and five strains (6%) of them MBL were positive as well as ESBL. Four strains (5%) were negative for both ESBL and MBL. Of the 24 MBL-producing strains, 23 strains (96%) were positive for MBL. All the 43 AmpC-producing strains were negative for both ESBL and MBL. Of the 156 ESBL- and MBL-nonproducing strains, 155 strains (99%) were negative for both ESBL and MBL, and one strain was positive for ESBL. With this method, the detection sensitivity was 95% and the specificity was 100% for ESBL, whereas the detection sensitivity was 96% and the specificity was 98% for MBL. These results were not significantly different from the results of the disc diffusion method. ConclusionThe DSTM method allows rapid detection of β-lactamases in 3 h and may be a useful replacement for the disc diffusion method.

Genomic Characterization of Carbapenem-Non-susceptible Pseudomonas aeruginosa Clinical Isolates From Saudi Arabia Revealed a Global Dissemination of GES-5-Producing ST235 and VIM-2-Producing ST233 Sub-Lineages.

Carbapenem-resistant P. aeruginosa has become a major clinical problem due to limited treatment options. However, studies assessing the trends in the molecular epidemiology and mechanisms of antibiotic resistance in this pathogen are lacking in Saudi Arabia. Here, we reported the genome characterization in a global context of carbapenem non-susceptible clinical isolates from a nationally representative survey. The antibiotic resistance profiles of the isolates (n = 635) collected over 14 months between March 2018 and April 2019 from different geographical regions of Saudi Arabia showed resistance rates to relevant β-lactams, aminoglycosides and quinolones ranging between 6.93 and 27.56%. Overall, 22.52% (143/635) of the isolates exhibited resistance to both imipenem and meropenem that were mainly explained by porin loss and efflux overexpression. However, 18.18% of resistant isolates harbored genes encoding GES (69.23%), VIM (23.07%), NDM (3.85%) or OXA-48-like (3.85%) carbapenemases. Most common GES-positive isolates produced GESs −5, −15 or −1 and all belonged to ST235 whereas the VIM-positive isolates produced mainly VIM-2 and belonged to ST233 or ST257. GES and VIM producers were detected at different sampling periods and in different surveyed regions. Interestingly, a genome-wide comparison revealed that the GES-positive ST235 and VIM-2-positive ST233 genomes sequenced in this study and those available through public databases from various locations worldwide, constituted each a phylogenetically closely related sub-lineage. Profiles of virulence determinants, antimicrobial resistance genes and associated mobile elements confirmed relatedness within each of these two different sub-lineages. Sequence analysis located the blaGES gene in nearly all studied genomes (95.4%) in the same integrative conjugative element that also harbored the acc(6′)-Ib, aph(3′)-XV, aadA6, sul1, tet(G), and catB resistance genes while blaVIM–2 in most (98.89%) ST233-positive genomes was co-located with aac(6′)-I1, dfrB-5, and aac(3′)-Id in the same class I integron. The study findings revealed the global spread of GES-5 ST235 and VIM-2 ST233 sub-lineages and highlighted the importance of routine detection of rare β-lactamases.

Antibiotic-functionalized gold nanoparticles for the detection of active β-lactamases.

Antimicrobial resistance (AMR) continues to threaten the effective treatment and prevention of bacterial infections. The spread of resistant infections is accelerated by the lack of fast and cost-effective tests for the detection of AMR at the point-of-care. We aimed to address this challenge by developing a diagnostic tool to detect one of the major forms of AMR, the β-lactamase enzymes. Antibiotic-functionalized gold nanoparticles (AuNPs) have been successfully developed for the detection of β-lactamases in challenging biological media, namely undiluted urine. Furthermore, these tools are compatible with samples containing a urine sample preservative (boric acid) or hematuria (blood). The functionalized AuNPs interact with the active β-lactamases, resulting in the hydrolysis of the surface-bound antibiotics, which then inhibits binding of the AuNPs to a capture protein (a penicillin-binding protein) to indicate the presence of active β-lactamases. We successfully integrated the antibiotic-functionalized AuNPs into a new lateral flow assay (LFA), which can be used to detect active β-lactamases down to the detection limit of 11 nM. While we demonstrate the use of antibiotic-functionalized AuNPs in an LFA format to provide a novel method of detecting active β-lactamases, these functionalized AuNPs are amenable to a range of alternative diagnostic technologies and could lead to vital point-of-care diagnostics for the early detection of multi-drug resistant infections.

Ultrasensitive detection of β-lactamase-associated drug-resistant bacteria using a novel mass-tagged probe-mediated cascaded signal amplification strategy.

The emergence and spread of drug-resistant bacteria (DRB) is a global health threat. Early and accurate detection of DRB is a critical step in the treatment of DRB infection. However, traditional assays for DRB detection are time-consuming and have inferior analytical sensitivity and quantification capability. Herein, a mass-tagged probe (MP-CMSA)-mediated enzyme- and light-assisted cascaded signal amplification strategy was developed for the ultrasensitive detection of β-lactamase (BLA), an enzyme closely associated with most DRB. Each MP-CMSA probe contained multiple poly(amidoamine) (PAMAM) dendrimer molecules immobilized on a streptavidin agarose bead via a BLA-cleavable linker, and each dendrimer was modified with multiple mass tags via a photo-cleavable linker. In BLA detection, BLA could cleave the BLA-cleavable linker, leading to dendrimers shedding from the MP-CMSA probe to achieve enzyme-assisted signal amplification. Then, each dendrimer can further release mass tags under UV light to achieve light-assisted signal amplification. After this cascaded signal amplification, the released mass tags were ultimately quantified by mass spectrometry. Consequently, the sensitivity of BLA detection can be significantly enhanced by four orders of magnitude with a detection limit of 50.0 fM. Finally, this approach was applied to the blood samples from patients with DRB. This platform provides a potential strategy for the sensitive, rapid and quantitative detection of DRB infection.

Detection and characterisation of extended-spectrum and plasmid-mediated AmpC β-lactamase produced by Escherichia coli isolates found at poultry farms in Bosnia and Herzegovina.

Extended-spectrum β-lactamases (ESBLs) hydrolyse extended-spectrum cephalosporins (ESC) and aztreonam. As ESBL-producing organisms have been identified in food producing animals, the aim of our study was to detect and analyse such Escherichia coli isolates from poultry. Antibiotic susceptibility of the isolates was determined with disk-diffusion and broth microdilution methods. ESBLs were detected with the double-disk synergy and inhibitor-based test with clavulanic acid. The transferability of cefotaxime resistance was determined with conjugation experiments, and genes encoding ESBLs, plasmid-mediated AmpC β-lactamases, and quinolone resistance determinants identified by polymerase chain reaction. The study included 108 faecal samples (cloacal swabs) from 25 different poultry farms in the Zenica-Doboj Canton, Bosnia and Herzegovina. Of these, 75 (69.4 %) were positive for E. coli, of which 27 were resistant to cefotaxime, amoxicillin, cefazoline, and cefriaxone, and susceptible to imipenem, meropenem, ertapenem, and amikacin. All 27 cefotaxime-resistant isolates were positive in double-disk synergy and combined disk tests. Eighteen isolates transferred cefotaxime resistance to E. coli recipient. Twenty-one isolates were positive for the blaCTX-M-1 cluster genes and seven for blaCTX-M-15. Fourteen were positive for the blaTEM genes. The most frequent plasmid incompatibility group was IncFIB, whereas IncFIA and Inc HI1 were present in only a few isolates. Two different sequence types (STs) were identified: ST117 and ST155. The emergence of ESBL-producing E. coli in farm animals presents a public health threat, as they can colonise the intestine and cause infections in humans.

A Prospective Study on Neonatal Sepsis in a Tertiary Hospital, Nepal

About 20 % of neonates develop sepsis and among them approximately 1% die due to sepsis-related causes. Bacterial pathogens are the commonest cause of neonatal sepsis which is either early-onset (&lt;72 hours of age) or late-onset (&gt;72 hours). Little is known about the epidemiology and antimicrobial susceptibility pattern of sepsis causing bacterial pathogens in Nepal. A prospective study was carried out among neonates suspected to have sepsis and admitted to Tribhuwan University Teaching Hospital from January to December 2016. Clinical suspicion of sepsis was made based on clinical findings and laboratory parameters, later confirmed by isolation of organisms in blood culture. Drug resistance pattern of Gram-positive and Gram-negative bacteria were studied by standard methods. Meropenem resistant Gram-negative bacteria were processed for the detection of β-lactamases and resistant genes were detected by X-pert Carba-R (Cepheid) Assays. Of 372 neonates with clinically suspected sepsis, 132 (35.4%) had blood culture positivity, with 47% early-onset and 53% late-onset sepsis. Coagulase-negative Staphylococcus aureus (CONS) was the most common (37.9%) etiological agent followed by Klebsiella pneumoniae (12.9%). Of all 132 isolates, 81 (61.3%) were Gram-positive of which 22 (27.2%) were multi-drug resistant (MDR), three (3.7%) were methicillin-resistant S. aureus (MRSA), and 14 (17.2%) were methicillin-resistant CoNS; and 50 (37.8%) were Gram-negative of which 26 (52%) were MDR and 29 (58%) were resistant to β-lactamases. The blaKPC gene was detected in four isolates of K. pneumoniae, two of E. coli, one ABC (Acinetobacter baumanii complex), and one Enterobacter aerogenes whereas blaNDM gene was detected in one isolate of K. pneumoniae, two of E. coli, two Pseudomonas aeruginosa, one Acinetobacter baumanii complex, and one Enterobacter aerogenes. Overall mortality due to sepsis-related causes was 7.6% (10 of 132). One-third of clinically suspected neonatal sepsis cases were culture positive. Late-onset sepsis was more common than early onset. CoNS was the predominant bacterial isolate followed by Klebsiella pneumoniae, with high rates of multi-drug resistance.

Accuracy of a Rapid Multiplex Polymerase Chain Reaction Plus a Chromogenic Phenotypic Test Algorithm for Detection of Extended-Spectrum β-Lactamase and Carbapenemase-Producing Gram-Negative Bacilli in Positive Blood Culture Bottles.

We studied the performance of an algorithm combining multiplex polymerase chain reaction with phenotypic detection of extended-spectrum β-lactamases and carbapenemases directly from positive blood culture bottles in patients with gram-negative bacteremia and found good concordance with routine cultures. Such an algorithm may be a tool to improve time to optimal therapy in patients with gram-negative bacteremia.

Antimicrobial resistance of Haemophilus influenzae isolates from pediatric hospitals in Mainland China: Report from the ISPED program, 2017–2019

PurposeThis study set out to determine the antimicrobial resistance trends of Haemophilus influenzae isolates from pediatric hospitals in Mainland China, which would provide basis for clinical treatment. MethodsThe Infectious Disease Surveillance of Pediatrics (ISPED) collaboration group conducted this study. H. influenzae strains isolated from nine pediatric hospitals in Mainland China were included. Disk diffusion method was used for antimicrobial susceptibility test. Cefinase disc was used for detection of β-lactamase. ResultsIn total, 13810 H. influenzae isolates were included during 2017–2019: 93.17% of which were from respiratory tract specimens, 4.63% from vaginal swabs, 1.10% from secretion, and 1.10% from others. Of all strains, 63.32% isolates produced β-lactamase; 8.22% isolates were β-lactamase-negative and ampicillin-resistant (BLNAR). The resistance to sulfamethoxazole-trimethoprim was 70.98%, followed by resistance to ampicillin (69.37%), cefuroxime (51.35%), ampicillin-sulbactam (38.82%), azithromycin (38.21%), amoxicillin-clavulanate (35.28%). More than 90% of H. influenzae isolates were susceptible to ceftriaxone, cefotaxime, meropenem, levofloxacin and chloramphenicol. The resistance rate of ampicillin and azithromycin in H. influenzae showed an increasing trend through the years. Statistically significant differences in antibiotic-resistance rates of all the antibiotics except chloramphenicol were found in different regions. The major Multi-Drug Resistance pattern was resistant to β-lactams, macrolides, and sulfonamides. ConclusionsThere is a rising trend of resistance rate of ampicillin and azithromycin in H. influenzae. Antimicrobial resistance of H. influenzae deserves our ongoing attention. Third-generation cephalosporin could be the preferred treatment option of infections caused by ampicillin-resistant H. influenzae.

A novel air-dried multiplex high-resolution melt assay for the detection of extended-spectrum β-lactamase and carbapenemase genes

This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended-spectrum β-lactamase (ESBL) (blaSHV and blaCTX-M groups 1 and 9) and carbapenemase (blaNDM, blaIMP, blaKPC, blaVIM and blaOXA-48-like) genes that confer resistance to cephalosporins and carbapenems. The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and the UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility. Assay reproducibility was evaluated across five different real-time quantitative PCR (qPCR) instruments [Rotor-Gene® Q, QuantStudioTM 5, CFX96, LightCycler® 480 and Magnetic Induction Cycler (Mic)]. Assay stability was also assessed under different storage temperatures (6.2 ± 0.9°C, 20.4 ± 0.7°C and 29.7 ± 1.4°C) at six time points over 8 months. The sensitivity and specificity (with 95% confidence intervals) for detecting ESBL and carbapenemase genes was 94.7% (92.5-96.5%) and 99.2% (98.8-99.5%) compared with the reference gel-based PCR and sequencing and 98.3% (97.0-99.3%) and 98.5% (98.0-98.9%) compared with the original HRM wet PCR mix format. Overall agreement was 91.1% (90.0-92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay, and no loss of sensitivity occurred under all storage conditions and time points. We present a ready-to-use air-dried HRM PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve antimicrobial resistance detection.

Resistance of Some Bacteria Isolated from Urinary Tract Infections in Elderly Patients to Antibiotics and Detection of β-lactamases in Them

Urinary tract infection is the second-largest infection, especially in elderly people, and the increasing use of antibiotics has led to the emergence and development of resistance in isolated and other bacteria in this study Fifty urine samples from elderly patients with urinary tract infections were collected from both sexes in Mosul city. Twenty-six bacterial isolates were isolated and diagnosed by ordinary bacteriological tests. The isolates were related to the species. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis. After isolation, their resistance to 14 selected antimicrobial agents was tested by the disk diffusion method. The presence of β-lactamases, in general, was detected by acidimetric method, the detection of extended-spectrum β-lactamases(ESBLs) was performed by double disk method, while the detection of AmpC β-lactamases was performed by cefoxitin resistance method. The isolates showed clear resistance to β-lactam antibiotics ranged (50%--92.3%),and to ciprofloxacin, gentamicin, sulfamethoxazole, streptomycin, rifampin with rates; (53.8%),(57.7%), (61.5%), (80.8%), (88.5%), respectively, while they showed reduced resistance to amikacin (15.4%). β-lactamases in general were detected in (96.1%) of the isolates, while (19.2%) of them gave a positive result for the detection of (ESBLs). AmpC β-lactamases were detected in (69.2%) of the isolates as they showed a clear resistance to cefoxitin which indicates that they are potential producers of these enzymes.

Detection of β-Lactamase–Producing Proteus mirabilis Strains of Animal Origin in Andhra Pradesh, India and Their Genetic Diversity

Proteus mirabilis is abundant in soil and water. Although this bacterium is part of the normal human intestinal flora, it can cause serious infections in humans, including complicated urinary tract infections. This pathogen is also commonly associated with multidrug resistance. In the present study, analysis of 1,093 samples from foods of animal origin and animal intestinal samples recovered 232 P. mirabilis isolates identified by PCR assay. Of these 232 isolates, 72 produced β-lactamase (determined by both phenotypic and genotypic methods), with the highest prevalence in poultry cloacal swabs (11.82%) followed by mutton (9.18%), khoa (6.32%), pork (5.63%), pig rectal swabs (5.52%), beef (5.45%), and chicken (5.13%) but none from sheep rectal swabs and bovine rectal swabs. Among β-lactamase genes, blaTEM was the predominant gene detected (59 isolates) followed by blaOXA (11 isolates), blaSHV (5 isolates), blaFOX (5 isolates), blaCIT (4 isolates), blaCTX-M1 and blaCTX-M9 (2 isolates each) and blaCTX-M2, blaDHA, and blaEBC (1 isolate each). None of the isolates carried blaACC, blaMOX, or carbapenemase genes (blaVIM, blaIMP, blaKPC, and blaNDM-1). Dendrogram analysis of enterobacterial repetitive intergenic consensus sequences and repetitive extragenic palindromic sequences obtained with PCR analysis of β-lactamase-producing isolates revealed 63 isolates, but 9 isolates did not yield bands. The analysis revealed that 6.58% of the samples had β-lactamase-producing P. mirabilis isolates that may affect food safety and contaminate the environment. Further genotyping revealed the genetic relationships between isolates of different origin. These findings emphasize the need for careful use of antibiotics to control the spread of β-lactamase-producing bacteria.