Nim Genes Research Articles

The emergence of metronidazole-resistant Prevotella bivia harboring nimK gene in Japan.

Metronidazole (MTZ) is an important antimicrobial agent in anaerobic infections and is widely used in clinical settings. The rate of MTZ resistance in anaerobic bacteria has been increasing in recent years, and the nim gene (nitro-imidazole reductase) is one of the resistance mechanisms. Prevotella bivia is found in humans in the urinary tract and vagina and is known to cause infections in some cases. One of the nim genes, nimK, has recently been discovered in this species of bacteria, but there are no reports of antimicrobial resistance (AMR)-related regions in its whole genome level. In this study, we analyzed the AMR region of nimK-positive P. bivia derived from clinical specimens based on comparisons with other anaerobic genomes. P. bivia was found to be engaged in horizontal gene transfer with other anaerobic bacteria, and the future spread of the nimK gene is a concern.

The phenotypic and genotypic antibiotic susceptibility of vaginal Lactobacillus with potential probiotic properties isolated from healthy women in northern Iran

Background and Objectives: This study assesses the antibiotic susceptibility of vaginal Lactobacillus strains and provides data for determining the prevalence of certain antibiotic resistance genes in the new strains of lactobacilli serving as probiot- ics and selected from healthy women in northern Iran. Materials and Methods: One hundred premenopausal non-pregnant women in the reproductive age range of 22-50 years participated in this study. The potential probiotic vaginal lactobacilli used in the study included Lactobacillus crispatus (34.2%), Lactobacillus gasseri (26.3%), Lactobacillus johnsonii (10.5%), Lactobacillus acidophilus (15.7%) and Lactoba- cillus jensenii (13.1%). The phenotypic antibiotic susceptibility of the strains was determined by E test and DNA extraction and PCR were performed to examine the antibiotic resistance genes. Results: 38 potential probiotic vaginal lactobacilli were isolated. All the strains of lactobacilli were resistant to metronida- zole and trimethoprim/sulfamethoxazole and all of the strains were susceptible to ampicillin and chloramphenicol antibiotics. The results showed that ermB, ermC, and ermA genes were observed in the strains of Lactobacillus acidophilus. Metronida- zole resistance (nim) gene was also found in one strain of Lactobacillus crispatus and Lactobacillus johnsonii. The amino- glycoside resistance (aac6'-aph2") gene was observed in 8% of the strains. Also, tetM, tetK and tetW genes were found in more than 80% of the Lactobacillus strains. Conclusion: The antimicrobial susceptibility of vaginal lactobacilli is an important criterion for establishing whether or not the organism is a probiotic. A high level of resistance to clinical antibiotics, such as metronidazole and aminoglycosides, was demonstrated. Antibiotic resistant genes also appeared widely in vaginal lactobacilli.

Proteomics-Based RT-qPCR and Functional Analysis of 18 Genes in Metronidazole Resistance of Bacteroides fragilis.

Previously, we reported that metronidazole MICs are not dependent on the expression levels of nim genes in B. fragilis strains and we compared the proteomes of metronidazole-resistant laboratory B. fragilis strains to those of their susceptible parent strains. Here, we used RT-qPCR to correlate the expression levels of 18 candidate genes in a panel of selected, clinical nim gene-positive and -negative B. fragilis strains to their metronidazole MICs. Metronidazole MICs were correlated with the expression of certain tested genes. Specifically, lactate dehydrogenase expression correlated positively, whereas cytochrome fumarate reductase/succinate dehydrogenase, malate dehydrogenase, phosphoglycerate kinase redox and gat (GCN5-like acetyltransferase), and relA (stringent response) regulatory gene expressions correlated negatively with metronidazole MICs. This result provides evidence for the involvement of carbohydrate catabolic enzymes in metronidazole resistance in B. fragilis. This result was supported by direct substrate utilization tests. However, the exact roles of these genes/proteins should be determined in deletion-complementation tests. Moreover, the exact redox cofactor(s) participating in metronidazole activation need to be identified.

First Report of Antibiotic Resistance Markers cfiA and nim Among Bacteroides fragilis Group Strains in Ecuadorian Patients.

In recent years, increasing resistance of Bacteroides fragilis to several antibiotics has been reported in different countries. The aim of this study was to evaluate the antibiotic resistance profiles of Bacteroides spp. isolated from clinical samples by phenotypic and molecular methods. A total of 40 nonrepetitive isolates of the B. fragilis group were studied from 2018 to 2019. The species was identified by API 20A system. The minimum inhibitory concentrations (MICs) were determined by Sensititre anaerobe MIC plate. The presence of the nim and cfiA genes was checked by conventional PCR. The association between genes and insertion sequence (IS) was performed by whole genome sequencing. Eleven isolates were categorized as metronidazole-resistant and only 2 isolates harbored the nim gene. Five isolates were imipenem-resistant, but cfiA gene was detected in two isolates. cfiA gene was closely related to the cfiA-4 allele and associated with IS614B. The nim gene was not related to any nim gene type and was considered a new variant named nimL. IS612 was found upstream of nimL gene. In view of the scarcity of data on B. fragilis, there is a need to surveil antibiotic resistance levels and molecular mechanisms to implement better antimicrobial therapies against this important group of bacteria.

Creep in nitroimidazole inhibitory concentration among the Entamoeba histolytica isolates causing amoebic liver abscess and screening of andrographolide as a repurposing drug

Infections by Entamoeba histolytica (E. histolytica) lead to considerable morbidity and mortality worldwide and treatment is reliant on a single class of drugs, nitroimidazoles. Treatment failures and intermittent reports of relapse from different parts of world indicate towards development of clinical drug resistance. In the present study, susceptibility testing of clinical isolates of E. histolytica was carried against metronidazole and tinidazole. Additionally, anti-amoebic property of active compounds of Andrographis paniculata was also evaluated. Prevalence of metronidazole resistance gene (nim) in patients attending hospital was also done to get comprehensive insight of present situation of drug resistance in E. histolytica. Mean inhibitory concentration 50 (IC50) value of E. histolytica isolates against metronidazole and tinidazole was 20.01 and 16.1 µM respectively. Andrographolide showed minimum mean IC50 value (3.06 µM). Significant percentage inhibition of E. histolytica isolates by andrographolide was seen as compared to metronidazole (p = 0.0495). None of E. histolytica isolates showed presence of nim gene. However, in stool samples from hospital attending population, prevalence of nimE gene was found to be 76.6% (69/90) and 62.2% (56/90) in diarrheal and non-diarrheal samples respectively. Inhibitory concentration of commonly used nitroimidazoles against clinical isolates of E. histolytica are on rise. Percentage inhibition of E. histolytica isolates by andrographolide was significantly higher than control drug metronidazole.

The effects of identical nim gene-insertion sequence combinations on the expression of the nim genes and metronidazole resistance in Bacteroides fragilis strains

In this study we examined whether the same nim gene-insertion sequence (IS) element combinations give rise to the same expression levels as they harbor shared IS element-borne promoters. From our quantitative analysis, we found that the expressions of the nimB and nimE genes with their cognate IS elements were similar, but the metronidazole resistance of these strains were more diverse.

Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis.

The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.

Phenotypic and Genotypic Correlation of Antimicrobial Susceptibility of Bacteroides fragilis: Lessons Learnt.

Background Bacteroides fragilis is an opportunistic pathogen causing severe infections, including bacteremia. There have been increased reports of antimicrobial resistance in B. fragilis. However, phenotypic testing of susceptibility is time consuming and not cost effective for anaerobes. The present study investigates the correlation of phenotypic susceptibility with genotypic markers; to determine if these could be considered for deciding empirical therapy for B. fragilis. Material and methods Bacteroides fragilis isolates from various clinical samples including exudates, tissue, and body fluids were collected between November 2018 and January 2020 in the Department of Clinical Microbiology, Christian Medical College (CMC) Vellore. Species identification was done by MatrixAssistedLaserDesorptionIonization time of flight mass spectrometry (MALDI TOF) according to the manufacturer's instructions. A total number of 51 B. fragilis isolates were tested against metronidazole, clindamycin, piperacillin/tazobactam, and meropenem phenotypically by agar dilution method using Clinical & Laboratory Standards Institute (CLSI) 2019 guidelines and minimum inhibitory concentrations (MIC) were interpretated. The genotypic markers for antimicrobial resistance genes (nim, emrF, and cfiA) were studied by polymerase chain reaction (PCR) assay as per the standard protocol on all isolates to detect resistance genes. Results B. fragilis isolates in this study expressed 45%, 41%, and 16% phenotypic resistance to clindamycin, metronidazole, and meropenem, respectively, with least resistance topiperacillin/tazobactam (6%). Among the metronidazole resistant isolates, 52% harbored nim gene. Nim gene was also present in 76% (23/30) of the metronidazole susceptible isolates. Similarly, cfiA was present in all eight meropenem resistant isolates in addition to 22% (9/41) of the susceptible isolates. All cfiA negative isolates were phenotypically susceptible. Interestingly, 74% (17/23) of the clindamycin resistant isolates were positive for ermF. Conclusions Detection of a limited set of genes does not always correlate with phenotypic resistance to metronidazole and clindamycindue to the reported influence of insertion sequence (IS) elements, efflux, and other genetic determinants. Certainly, the absence of the cfiA gene can be employed to rule out meropenem resistance. However, redundant use of antibiotics such as meropenem along with metronidazole could be avoided for B. fragilis, which might otherwise elevate meropenem resistance. Recommendation of metronidazole requires prior phenotypic testing due to the reported 41% resistance.

Molecular study on metronidazole resistance in Bacteroides fragilis group isolates from a South Indian tertiary care center

ObjectiveBacteroides species are an important part of human intestinal microbiota. They can cause infections of significant mortality and morbidity when moved out of their niche in the gut. The cornerstone drug for prophylaxis and therapy, metronidazole, is exhibiting signs of resistance, which are frequently attributed to nitroimidazole (nim) resistance genes. The aim of this study was to use Epsilometer test (E-test) to assess the metronidazole susceptibility and conventional PCR methodology to map the distribution of nim genes in Bacteroides fragilis group (BFG) isolates. MethodsMALDI-TOF MS was used to identify BFG isolates. Using the E-test methodology, metronidazole minimum inhibitory concentrations (MICs) were determined. The presence of nim genes in these isolates were checked by conventional PCR methodology. Sequencing was done on selected amplicons for determining the nim gene types. ResultsBacteroides fragilis accounted for 55.3% of the total 273 BFG members identified. Of these, 196 (71.8%) were susceptible, 43 (15.8%) intermediate and 34 (12.5%) resistant to metronidazole as determined by the E-test. nim gene was present in 101 (37%) of the total 273 isolates. Out of the 34 phenotypically resistant isolates (MIC ≥32 μg/ml), 29 harboured nim gene (Chi-square test, p < 0.0000001) but nim gene was absent in 5 (14.7%) isolates. Also, nim gene was detected in 72 (30.1%) of the 239 isolates with susceptible and intermediate metronidazole MIC. Sequencing of 20 amplicons gave a nimE gene type. ConclusionsIn view of the rising metronidazole resistance among BFG and its close association with nim genes, there is a need for implementing routine metronidazole susceptibility testing and more researches are needed to find the molecular basis of these nim genes.

Identification of cfxA gene variants and susceptibility patterns in β-lactamase-producing Prevotella strains

ObjectivesAntimicrobial-resistant isolates of Prevotella species, especially those resistant to β-lactams, have become increasingly common. Here, we aimed to elucidate the underlying mechanisms contributing to the emergence and spread of antimicrobial resistance in Prevotella species. MethodsPrevotella species were isolated from a variety of clinical specimens. β-lactamase production was determined using nitrocefin discs, and the determination of minimum inhibitory concentration (MIC) to ten antimicrobials was done by the agar dilution method. Four resistance genes (cfxA, tetQ, ermF, and nim) and cfxA-flanking regions were detected using polymerase chain reaction. cfxA and the flanking regions were sequenced, and a phylogenetic tree was constructed based on CfxA amino acid sequences using the UPGMA method. ResultsAmong the 45 Prevotella isolates identified, 35 (77.8%) produced β-lactamases and had the cfxA genes. The tetQ, ermF, and nim genes were detected in 53.3%, 17.8%, and 0% of the 45 isolates, respectively. Among the 33 sequenced cfxA alleles, cfxA2 (45.5%) was the most frequent, followed by cfxA3 (42.4%) and a novel variant (cfxA7, 12.1%). The novel CfxA7 β-lactamase had a novel L155F substitution not previously reported in CfxA variants. The MICs of all β-lactam agents tested, excluding cefmetazole and meropenem, were lower among cfxA7-positive isolates than in cfxA2-and cfxA3-positive isolates. ConclusionsDifferences in MICs of penicillins and cephalosporins may be due to amino acid substitutions in the CfxA variants, CfxA2, CfxA3, and CfxA7, among Prevotella isolates. Possession of cfxA-mobA, tetQ, and ermF may increase the risks of the emergence and spread of multidrug-resistant Prevotella species.

Prevalence of antimicrobial resistant genes in Bacteroides spp. isolated in Oita Prefecture, Japan

IntroductionBacteroides spp. are the most common anaerobic bacteria isolated from the human gastrointestinal tract. Several resistant genes are present in Bacteroides spp. However, most studies have focused on the prevalence of the cfiA gene in Bacteroides fragilis alone. We assessed the susceptibility to antimicrobial agents and the prevalence of cepA, cfiA, cfxA, ermF, nim, and tetQ genes in Bacteroides strains isolated from clinical specimens in our hospital. MethodsWe isolated 86 B. fragilis and 58 non-fragilis Bacteroides strains from human clinical specimens collected from January 2011 to November 2021. Resistance against piperacillin (PIPC), cefotaxime (CTX), cefepime (CFPM), meropenem (MEPM), clindamycin, and minocycline was determined. ResultsThe resistant rates of penicillins and cephalosporins in non-fragilis isolates were significantly higher than those in B. fragilis isolates. In B. fragilis isolates, the resistant rates of PIPC, CTX, and CFPM in cfxA-positive isolates were significantly higher than those in cfxA-negative isolates (71% vs. 16%, 77% vs. 19%, and 77% vs. 30%, respectively). Thirteen B. fragilis isolates harbored the cfiA gene, two of which were resistant to MEPM. Six of the 13 cfiA-positive B. fragilis isolates were heterogeneously resistant to MEPM. ConclusionIt is important to evaluate the use of MEPM as empirical therapy for Bacteroides spp. infections, considering the emergence of carbapenem resistance during treatment, existence of MEPM-resistant strains, and heterogeneous resistance.

Initial expression levels of nimA are decisive for protection against metronidazole in Bacteroides fragilis

ObjectivesIn the genus Bacteroides, the nim genes are resistance determinants for metronidazole, a nitroimidazole drug widely used against anaerobic pathogens. The Nim proteins are considered to act as nitroreductases. However, data from several studies suggest that the expression levels of Nim do not increase with increasing resistance which is conflicting with this notion. The impact of Nim protein levels on low-level metronidazole resistance, however, representing the early stage of induced resistance in the laboratory, has not been assessed as yet. MethodsThe nimA gene was cloned into two different plasmids and introduced into B. fragilis strain 638R. Expression levels of nimA mRNA were measured by RT-qPCR and compared to those in strain 638R harbouring plasmid pI417, the original clinical plasmid harbouring IS element IS1168 with the nimA gene. Further, metronidazole susceptibility was assessed by Etest and the activity of pyruvate:ferredoxin oxidoreductase (PFOR) was measured in all strains after induction of high-level metronidazole resistance. ResultsThe level of protection against metronidazole by nimA correleated with the level of expression of nimA mRNA. Further, the activity of PFOR in highly-resistant B. fragilis 638R was only preserved when expression levels of nimA were high. ConclusionsAlthough the development of high-level metronidazole resistance in B. fragilis strains with a nimA gene is not caused by an increase of nimA expression as compared to the less resistant parent strains, nimA expression levels might be of decisive importance in the early stage of resistance development. This has potential implications for metronidazole resistance in clinical isolates.

Modulation of Iron Import and Metronidazole Resistance in Bacteroides fragilis Harboring a nimA Gene

Bacteroides fragilis is a commensal of the human gut but can also cause severe infections when reaching other body sites, especially after surgery or intestinal trauma. Bacteroides fragilis is an anaerobe innately susceptible to metronidazole, a 5-nitroimidazole drug that is prescribed against the majority of infections caused by anaerobic bacteria. In most of the cases, metronidazole treatment is effective but a fraction of B. fragilis is resistant to even very high doses of metronidazole. Metronidazole resistance is still poorly understood, but the so-called nim genes have been described as resistance determinants. They have been suggested to encode nitroreductases which reduce the nitro group of metronidazole to a non-toxic aminoimidazole. More recent research, however, showed that expression levels of nim genes are widely independent of the degree of resistance observed. In the search for an alternative model for nim-mediated metronidazole resistance, we screened a strain carrying an episomal nimA gene and its parental strain 638R without a nim gene for physiological differences. Indeed, the 638R daughter strain with the nimA gene had a far higher pyruvate-ferredoxin oxidoreductase (PFOR) activity than the parental strain. High PFOR activity was also observed in metronidazole-resistant clinical isolates, either with or without a nim gene. Moreover, the strain carrying a nimA gene fully retained PFOR activity and other enzyme activities such as thioredoxin reductase (TrxR) after resistance had been induced. In the parental strain 638R, these were lost or very strongly downregulated during the development of resistance. Further, after induction of high-level metronidazole resistance, parental strain 638R was highly susceptible to oxygen whereas the daughter strain with a nimA gene was hardly affected. Ensuing RT-qPCR measurements showed that a pathway for iron import via hemin uptake is downregulated in 638R with induced resistance but not in the resistant nimA daughter strain. We propose that nimA primes B. fragilis toward an alternative pathway of metronidazole resistance by enabling the preservation of normal iron levels in the cell.

PREVOTELLA BIVIA BACTEREMIA POST TOCILIZUMAB IN A PATIENT WITH SEVERE COVID-19 PNEUMONIA

TOPIC: Chest Infections TYPE: Medical Student/Resident Case Reports INTRODUCTION: Anaerobes generally constitute a major component of the gut microbiome. They play a significant role in the pathogenesis of infections following disruption of mucosal integrity. Anaerobic organisms are usually difficult to grow and identify and hence may often be overlooked. We present a patient with severe COVID-19 pneumonia who developed de-novo Prevotella bivia bacteremia, following tocilizumab. CASE PRESENTATION: A 38 year old Caucasian male with morbid obesity and no other past medical history presented to the emergency department with complaints of cough, fever, chills and poor appetite for the past 10 days. Upon arrival, he was profoundly hypoxic with Spo2 70% on ambient air. He tested positive for COVID-19. On physical examination, his BMI was 62 kg/m2, he was febrile, in moderate respiratory distress, cardiopulmonary examination revealed coarse bilateral breath sounds. His physical examination was otherwise unremarkable. His chest radiograph demonstrated hazy opacification in both mid lungs and left lung base suggestive of multifocal pneumonia. CT chest did not reveal evidence of pulmonary embolism. Lower extremity ultrasound was however negative for a DVT. Laboratory findings was significant for elevated C-reactive protein 12.4 mg/dl. Patient was placed on heated high flow nasal cannula and received dexamethasone and tocilizumab. His respiratory status continued to worsen and he was subsequently intubated. Lower respiratory culture was positive for MSSA and patient was started cefepime and vancomycin was added as he clinically continued to decompensate. Within the next week, blood culture was positive for Prevotella bivia and his antibiotic regimen was changed to pipericillin-tazobactam. Repeat blood culture continued to be positive and this time, for Enterococcus faecalis. The antibiotic regimen was further changed to meropenem. Interestingly, on his physical examination, there was no abnormalities on his abdomen or pelvic area. CT abdomen pelvis did not shows any acute abnormalities. Following meropenem initiation, his subsequent blood cultures have been negative for any growth. Patient remains intubated with a guarded prognosis. DISCUSSION: Prevotella bivia is one of several species of bacteria in the genus Prevotella. It is an anaerobic gram negative organism that is found in the oral, vaginal, and gut microbiome. Some of the infections caused by this organism include aspiration pneumonia, lung abscess, otitis media, and sinusitis. Various species of Prevotella have been isolated from abscesses and burns close of the mouth, bite, paronychia, urinary tract infection, brain abscess, osteomyelitis, and bacteremia when there is an associated respiratory tract infections. Prevotella bivia bacteremia is highly uncommon. CONCLUSIONS: As we continue to learn more about COVID-19 infection and its treatment modalities, we may come across such unusual scenarios. REFERENCE #1: 27.Curtis JR, Perez-Gutthann S, Suissa S, et al. Tocilizumab in rheumatoid arthritis: a case study of safety evaluations of a large postmarketing data set from multiple data sources. Semin Arthritis Rheum 2015;44:381. REFERENCE #2: Yanagisawa M, Kuriyama T, Williams DW, Nakagawa K, Karasawa T. Proteinase activity of prevotella species associated with oral purulent infection. Curr Microbiol. 2006;14(5):375–378. doi: 10.1007/s00284-005-0261-1 REFERENCE #3: Metronidazole Resistance in Prevotella spp. and Description of a New nim Gene in Prevotella baroniaeC. Alauzet, F. Mory, C. Teyssier, H. Hallage, J. P. Carlier, G. Grollier, A. Lozniewski DISCLOSURES: No relevant relationships by Alfred Aiyanyor, source=Web Response No relevant relationships by Navitha Ramesh, source=Web Response No relevant relationships by Q. Kamran Uddin, source=Web Response

Factors associated with high rates of recurrence of amebic liver abscess (ALA) in north India

Recurrence of amebic liver abscess (ALA), once considered unusual, is increasingly being reported, despite proper management. Realizing the endemicity of ALA in the study setup, this 2-year follow-up study was conducted to investigate the recurrent cases and study the associated factors. A total of 101 confirmed cases of ALA were followed up for a period of 2 years. Recurrent cases were studied for associated bacterial flora, presence of resistance genes (nim), level of matrix metalloproteinase 3 and MMP-9, and genotypes of Entamoeba histolytica and statistically compared with the nonrecurrent cases as controls. Recurrence rates of 8.9% (nine patients) were detected. The presence of Prevotella along with an increased level of MMP-9 in abscess fluid and large size of abscesses (11 × 10.8 cm) was found to be significantly associated with recurrence in ALA. Among the nine cases, the presence of nimE gene was detected in two (22.2%) patients. The genotyping of E. histolytica strains showed that in seven (77.7%) cases, the genotype of E. histolytica was the same in the primary and recurrent samples. This study reports a high rate of recurrence in the cases of ALA, hinting toward the gradual development of clinical resistance toward the commonly used drug. The presence of nim gene and Prevotella in abscess fluid along with increased MMP-9 levels and large abscess size could be important predictors of recurrent ALA.

Antibiotic Resistance in Bacteroides and Prevotella to β-Lactams, Lincosamide and Nitroimidazole: A 20 Year Survey in Lagos, Nigeria

The propensity to develop resistance to antibiotics has accounted for the predominance of Bacteroides and Prevotella in infections due to anaerobic bacteria. The observed differences in resistance pattern across geographical boundaries underscored the timeliness for this study to review data on antibiotic susceptibility and resistance markers amongst Bacteroides and Prevotella to β-lactams, lincosamide and nitroimidazole from selected hospitals in Lagos, Nigeria from 1992-2011. Bacteroides, mostly B. fragilis were the sole anaerobic gram negative bacilli in peritonitis following lower abdominal surgery and bloodstream infections, while Prevotella bivia and P. melaninogenica were more frequent in periodontal abscess, pelvic inflammatory disease and chronic suppurative otitis media. The MIC range and MIC90 of the anaerobes for the antibiotics does not indicate changes in group resistance to antibiotics though isolated cases were recorded. While clear cut patterns were not established for other species, B. fragilis resistance increased for amoxicillin-clavulanic acid from 18.2% in 1992-1996 to 31.4% in 2006-2011 and from 27.3% in 1992-1996 to 34.3% in 2006-2011 for cefoxitin. In contrast, decrease susceptibility was obtained against clindamycin (54.5% in 1992-1996 to 22.9% in 2006-2011). Similarly, 22.7% of B. fragilis strains have the cepA and/or cfxA gene in 1992-1996 compared to 32.1% in 2006-2011. While not all isolates with cepA and/or cfxA genes were resistant to the β-lactams, all isolates carrying the ermF or nim genes were resistant to the lincosamide (clindamycin) or nitroimidazole (metronidazole) respectively. The presence of antibiotics resistance genes can be used in tracking resistance amongst species of Bacteroides and Prevotella. With emerging new technologies, gene screening may prove more effective and reliable for therapeutic decisions for anaerobic bacterial infections especially since the genes can be screened from the infective exudates. This will eliminate the need for culture and antibiotic susceptibility testing for this group of fastidious microorganisms.

Phenotypic and genotypic antimicrobial resistance in clinical anaerobic isolates from India.

BackgroundAntimicrobial resistance (AMR) in anaerobes remains a neglected field. The laborious procedures, non-compliance with the standard methodology and differences in interpretive breakpoints add variation in resistance data.ObjectivesTo assess the phenotypic and genotypic resistance among clinically important anaerobes to six antibiotics frequently used as empirical therapy for anaerobic infections.MethodsA total of 150 anaerobic isolates were recovered from clinical specimens. The antimicrobial susceptibility was determined by the breakpoint agar dilution method as per CLSI guidelines. The presence of genes encoding resistance to metronidazole (nim gene), imipenem (cfiA gene) and mobilizable insertion sequence (IS) elements was detected to comprehend their association with phenotypic resistance.ResultsThis is a first study of its kind from the Indian subcontinent looking at the AMR and associated genes in anaerobes. Resistance to metronidazole, clindamycin, imipenem, piperacillin/tazobactam and cefoxitin was 32.6%, 42.6%, 0.6%, 38% and 35.3%, respectively. No resistance was observed to chloramphenicol. The nim gene was detected in 24.6% of isolates, of which 70.2% were resistant by phenotype. On sequencing, the PCR products of six random nim genes showed a close similarity to nimE of Bacteroides fragilis with 99% nucleotide and 100% amino acid sequence similarity. The cfiA gene, associated with imipenem resistance, was detected in 16% of isolates.ConclusionsThe possibility of isolates carrying AMR genes to become resistant to antibiotics by acquisition of IS elements mandates attention to periodically monitor the resistance patterns and geographic distribution of these genes and IS elements to understand the trends of AMR in anaerobes.

Sequencing of Clostridium perfringens toxin genes (cpa, etx, iap) from Iraqi hospitals and detection by PCR of the genes encoding resistance to metronidazole, tetracycline, and clindamycin

PurposeThis cross sectional study was designed to investigate the molecular detection of C. perfringens toxins (alpha, epsilon, iota) and antibiotics resistance genes, as well as sequencing of their toxin genes. MethodsDifferent wound swabs were taken from 140 patients. PCR was applied for detection of clostridial toxins; alpha toxin (cpa) gene, epsilon toxin (etx) gene, and iota toxin (iap) gene. Metronidazole nim gene, tetracycline resistance genes; (tetQ, tetM, tetB, and tetW) and clindamycin resistance genes erm (A,B), were used for genotypic detection of antibiotic resistance. ResultsOut of 140 clinical samples collected, 7 isolates were detected using specific primer 16S–23S intergenic rRNA spacer gene of C. perfringens. Results showed presence of alpha toxin (cpa) genes in all clostridial isolates, Epsilon toxin (etx) genes in 2/7(28.4%), and iota toxin (iap) genes in 2/7 (28.4%). Results of antibiotic resistance genes showed that all isolates were not able to produce nim, tet W and Q, tet B, erm (A), erm (B) genes except for tetM. Gene sequence analysis for cpa gene showed that there were 3 mutations in the sample of this gene, the results of etx gene when had been sent 2 samples from this gene. The first sample showed that there was one mutation and the results of iap gene showed that there were no mutation in 2 samples of the iap gene. The samples had been showed identical 100%. ConclusionThe DNA sequence analysis of bacterial genome revealed several important feature including that may allow to confirm new the presence of toxin and to identify new or mutant toxins that may be missed by diagnostic PCR specifically target toxin encoding genes.

Diversity of antimicrobial resistance genes in Bacteroides and Parabacteroides strains isolated in Germany

ObjectivesBacteroides spp. are normal constituents of the human intestinal microflora, but they are also able to cause severe diseases. The aim of this study was to determine the diversity of antibiotic resistance genes found in phenotypically resistant Bacteroides and Parabacteroides strains. MethodsA total of 71 phenotypically resistant Bacteroides spp. from human clinical specimens were screened for the antibiotic resistance genes cfiA, tetQ, tetM, tet36, cepA, cfxA, nim, ermG, ermF, bexA, blaVIM, blaNDM, blaKPC, blaOXA-48 and blaGES. The presence of these genes was compared with phenotypic resistance to ampicillin/sulbactam, cefoxitin, ceftolozane/tazobactam, piperacillin/tazobactam, imipenem, meropenem, meropenem/vaborbactam, clindamycin, moxifloxacin, tigecycline, eravacycline and metronidazole. ResultstetQ was the most frequently detected gene, followed by cfiA, ermF, cfxA, ermG, cepA, nim and bexA. None of the strains were positive for tetM, tet36, blaVIM, blaNDM, blaKPC, blaOXA-48 or blaGES. Resistance to the tested β-lactams was mainly linked to the presence of the cfiA gene. Clindamycin resistance correlated with the presence of the genes ermG and ermF. The bexA gene was found in six strains, but only two of them were resistant to moxifloxacin. Tigecycline and eravacycline showed good activities despite the frequent occurrence of tetQ. The nim gene was detected in six isolates, five of which were resistant to metronidazole. ConclusionThe findings of our study support the general belief that antimicrobial resistance within Bacteroides should be taken into consideration. This underlines the necessity of reliable routine antimicrobial susceptibility test methods for anaerobic bacteria and the implementation of antimicrobial surveillance programmes worldwide.

Factors Associated with High Rates of Recurrence of Amebic Liver Abscess (ALA) in North India.

Recurrence of amebic liver abscess (ALA), once considered unusual, is increasingly being reported, despite proper management. Realizing the endemicity of ALA in the study setup, this 2-year follow-up study was conducted to investigate the recurrent cases and study the associated factors. A total of 101 confirmed cases of ALA were followed up for a period of 2 years. Recurrent cases were studied for associated bacterial flora, presence of resistance genes (nim), level of matrix metalloproteinase 3 and MMP-9, and genotypes of Entamoeba histolytica and statistically compared with the nonrecurrent cases as controls. Recurrence rates of 8.9% (nine patients) were detected. The presence of Prevotella along with an increased level of MMP-9 in abscess fluid and large size of abscesses (11 × 10.8 cm) was found to be significantly associated with recurrence in ALA. Among the nine cases, the presence of nimE gene was detected in two (22.2%) patients. The genotyping of E. histolytica strains showed that in seven (77.7%) cases, the genotype of E. histolytica was the same in the primary and recurrent samples. This study reports a high rate of recurrence in the cases of ALA, hinting toward the gradual development of clinical resistance toward the commonly used drug. The presence of nim gene and Prevotella in abscess fluid along with increased MMP-9 levels and large abscess size could be important predictors of recurrent ALA.