Plasmid evolution in carbapenemase‐producing Enterobacteriaceae : a review

Please read abstract in the article.Table S1. Metadata of plasmids deposited at GenBank and included in this study.Supplementary dataset. Nucleotide sequences of plasmids included in this study and obtained from Genbank.https://nyaspubs.onlinelibrary.wiley.com/journal/174966322020-12-01hj2019Medical Microbiolog

Global epidemiology, genetic environment, risk factors and therapeutic prospects of MCR genes : a current and emerging update

BACKGROUND : Mobile colistin resistance (mcr) genes modify Lipid A molecules of the lipopolysaccharide, changing the overall charge of the outer membrane. RESULTS AND DISCUSSION: Ten mcr genes have been described to date within eleven Enterobacteriaceae species, with Escherichia coli, Klebsiella pneumoniae, and Salmonella species being the most predominant. They are present worldwide in 72 countries, with animal specimens currently having the highest incidence, due to the use of colistin in poultry for promoting growth and treating intestinal infections. The wide dissemination of mcr from food animals to meat, manure, the environment, and wastewater samples has increased the risk of transmission to humans via foodborne and vectorborne routes. The stability and spread of mcr genes were mediated by mobile genetic elements such as the IncHI2 conjugative plasmid, which is associated with multiple mcr genes and other antibiotic resistance genes. The cost of acquiring mcr is reduced by compensatory adaptation mechanisms. MCR proteins are well conserved structurally and via enzymatic action. Thus, therapeutics found effective against MCR-1 should be tested against the remaining MCR proteins. CONCLUSION: The dissemination of mcr genes into the clinical setting, is threatening public health by limiting therapeutics options available. Combination therapies are a promising option for managing and treating colistin-resistant Enterobacteriaceae infections whilst reducing the toxic effects of colistin.The National Health Laboratory Service (NHLS) and the National Research Foundation.https://www.frontiersin.org/journals/cellular-and-infection-microbiologydm2022Medical Microbiolog

Current and emerging polymyxin resistance diagnostics : a systematic review of established and novel detection methods

The emergence of polymyxin resistance, due to transferable mcr genes, threatens public and animal health as there are limited therapeutic options. As polymyxin is one of the last-line antibiotics, there is a need to contain the spread of its resistance to conserve its efficacy. Herein, we describe current and emerging polymyxin resistance diagnostics to inform faster clinical diagnostic choices. A literature search in diverse databases for studies published between 2016 and 2020 was performed. English articles evaluating colistin resistance methods/diagnostics were included. Screening resulted in the inclusion of 93 journal articles. Current colistin resistance diagnostics are either phenotypic or molecular. Broth microdilution is currently the only gold standard for determining colistin MICs (minimum inhibitory concentration). Phenotypic methods comprise of agar-based methods such as CHROMagar™ Col-APSE, SuperPolymyxin, ChromID® Colistin R, LBJMR and LB medium; manual MIC-determiners viz., UMIC, MICRONAUT MIC-Strip and ComASP Colistin; automated antimicrobial susceptibility testing systems such as BD Phoenix, MICRONAUT-S, MicroScan, Sensititre and Vitek 2; MCR-detectors such as lateral flow immunoassay (LFI) and chelator-based assays including EDTA-and DPA-based tests, that is, combined disk test, modified colistin broth-disk elution (CBDE), Colispot, and Colistin MAC test as well as biochemical colorimetric tests, that is, Rapid Polymyxin NP test and Rapid ResaPolymyxin NP test. Molecular methods only characterize mobile colistin resistance; they include PCR, LAMP and whole-genome sequencing. Due to the faster turnaround time (≤3 h), improved sensitivity (84%–100%) and specificity (93.3%–100%) of the Rapid ResaPolymyxin NP test and Fastinov®, we recommend this test for initial screening of colistin-resistant isolates. This can be followed by CBDE with EDTA or the LFI as they both have 100% sensitivity and a specificity of ≥94.3% for the rapid screening of mcr genes. However, molecular assays such as LAMP and PCR may be considered in well-equipped clinical laboratories.We hereby regretfully report the death of our colleague Professor Nontombi Marylucy Mbelle, who died during the submission of this article. This work is dedicated to her memory.The National Health Laboratory Service (NHLS)http://wileyonlinelibrary.com/journal/jamam2022Medical Microbiolog

Antimicrobial susceptibility and serotype distribution of Streptococcus agalactiae rectovaginal colonising isolates from pregnant women at a tertiary hospital in Pretoria, South Africa : an observational descriptive study

BACKGROUND. Streptococcus agalactiae or group B streptococcus (GBS) is a significant cause of neonatal sepsis. Intrapartum antibiotic prophylaxis is recommended for pregnant women identified to be rectovaginally colonised between 34 and 37 weeks’ gestational age to decrease the risk of invasive disease in their newborns. An effective multivalent GBS vaccine may prevent a broader scope of GBS-associated diseases, such as GBS early-onset disease, GBS late-onset disease, spontaneous abortion, stillbirth and maternal bacteraemia. Serotype distribution of GBS isolates is essential to determine the efficacy of such a vaccine. OBJECTIVES. To investigate serotype distribution and antimicrobial susceptibility patterns of GBS isolates cultured from rectovaginal specimens during pregnancy. METHODS. Sixty-nine archived maternal colonising isolates were tested against penicillin, erythromycin, clindamycin, vancomycin and levofloxacin. Minimum inhibitory concentration testing was performed using the ETEST method. Serotyping was performed by the latex agglutination method. RESULTS. The most common serotypes detected were Ia (54%), III (20%), V (16%), II (6%), IV (2%) and Ib (1%). All isolates were fully susceptible to penicillin, vancomycin and levofloxacin. Eight (11%) and 50 (56%) isolates showed intermediate resistance to erythromycin and clindamycin, respectively, and 1 isolate was resistant to erythromycin. The macrolide-lincosamide-streptogramin B (MLSB) phenomenon was noted in 3 (4%) of the isolates. CONCLUSIONS. GBS-colonising isolates remain susceptible to penicillin, which remains the drug of choice for intrapartum antibiotic prophylaxis and treatment of invasive disease in newborns. Macrolides should only be used if clinically indicated due to the high prevalence of intermediate resistance. A pentavalent GBS vaccine currently in phase I trials should provide coverage for 97% of the isolates identified in this study.The National Health Laboratory Service (NHLS) and the Respiratory and Meningeal Pathogens Research Unit, University of the Witwatersrand, Johannesburg.http://www.samj.org.zaam2021Medical Microbiolog

Comparison of Xpert GBS v. culture for rapid detection of group B streptococcus in pregnant women : sensitivity, specificity and predictive values

BACKGROUND. Group B streptococcus (GBS) is a leading cause of invasive disease, particularly in newborns. Seventy-five percent of neonates will be colonised by mothers carrying the organism. Confirmation of maternal colonisation with GBS is essential for prompt treatment and prevention of neonatal sepsis. The current gold standard of culture for isolation of GBS has a disadvantage of long turnaround time (24 - 72 hours). Rapid assays are required to determine maternal carriage of GBS. OBJECTIVES. To determine the usefulness of the Xpert GBS technology v. culture methods to detect GBS carriage in pregnant women. METHODS. This was a prospective observational study of 284 pregnant women between 26 and 37 weeks’ gestation. Two vaginorectal swabs were collected from each participant. One swab was processed using the gold-standard culture method, while the second swab was processed using the Xpert GBS assay. The performance of the Xpert GBS assay was then compared with that of the culture method. RESULTS. Two swabs were processed from each of 284 pregnant women between 26 and 37 weeks’ gestation. Culture detected 70 GBS isolates from a total of 279 specimens (25.1%), whereas the Xpert GBS detected 66 positive specimens (23.7%). The Xpert GBS assay had a sensitivity of 87% and specificity of 98%, with a positive predictive value of 92% and a negative predictive value of 96%. CONCLUSIONS. The Xpert GBS assay is a rapid and sensitive tool for prenatal detection of GBS. The assay should ideally be available in every labour ward, where women can be screened for GBS on arrival.The research described in this article was an MMed project of MS, the main author.A National Health Laboratory Services Research Grant (no. 94443) in fulfilment of MS’s MMed project.http://www.samj.org.zaam2019Medical MicrobiologyObstetrics and Gynaecolog

A comparative evaluation of the new Genexpert MTB/RIF ultra and other rapid diagnostic assays for detecting tuberculosis in pulmonary and extra pulmonary specimens

Studies evaluating the new GeneXpert Ultra with other rapid diagnostic assays are limited, particularly in diferent geographical settings. The performance of the GeneXpert Ultra, the GeneXpert G4, the Line probe assays (LPA) and auramine smear microscopy in detecting TB in pulmonary and extrapulmonary samples were thus evaluated. Remnants (n=205 samples) of pulmonary (n=125 samples) and extra-pulmonary (n=80 samples) specimens from TB suspects were prospectively collected. Each sample was divided for diagnosis using microscopy, GeneXpert MTB/RIF assays, and LPA; these were all comparatively evaluated, using the MGIT 960 culture as a gold standard. The sensitivity and specifcity of microscopy, Xpert Ultra, Xpert G4 and MTBDRplus (ver 2) in pulmonary samples were respectively: 82.00% and 90.28%; 88.00% and 58.57%; 79.59% and 90.28%; 80.00% and 11.11%. For extrapulmonary specimen, the sensitivity and specifcity were respectively: 53.85% and 98.51%; 69.23% and 49.25%; 50.00% and 97.01%; 69.23% and 25.37%. The new and improved GeneXpert Ultra assay was more sensitive than GeneXpert G4 and LPA in both pulmonary and extra pulmonary samples, albeit with lower specifcity than the GeneXpert G4. The auramine and LPA tests were also highly sensitive, although the LPA was less specifc.University of Pretoria; National Research Foundation; Medical Research Council of South Africahttps://www.nature.com/sreppm2020Medical Microbiolog

Molecular characteristics and genotypic diversity of enterohaemorrhagic Escherichia coli O157:H7 isolates in Gauteng region, South Africa

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is one of the major foodborne and waterborne pathogens causing severe diseases and outbreaks worldwide. There is scarcity of EHEC O157:H7 data in South Africa. This study was carried out to determine the molecular characteristics and genotypic diversity of EHEC O157:H7 isolates in the Gauteng region, South Africa. Samples were cultured on selective chromogenic media. Antibiotic susceptibility profile of isolates was determined using the VITEK®-2 automated system. Isolates were characterised using multiplex PCR assays and the genetic diversity was determined using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 520 samples of which 270 environmental water samples and 250 stool specimens were collected and analysed. Overall, EHEC O157:H7 was recovered from 2.3% (12/520) of samples collected. Environmental water samples and clinical stool specimens showed a prevalence of 4.07% (11/270) and 0.4% (1/250) respectively. Antibiotic susceptibility profile varied from isolates with full susceptibility to isolates with resistance to multiple antibiotics. Most resistance was detected to the penicillins, specifically ampicillin (7/12), amoxicillin (3/12) and piperacillin/Tazobactam (3/12) followed by one of the folate inhibitors, trimethoprim (3/12) and the carbapenems, imipenem and meropenem (2/12) each. Three isolates harboured a combination of Shiga-toxins (Stx)-2, intimin (eae) and enterohaemolysin (hlyA) genes, while two isolates harboured the Stx-1, Stx-2 and hlyA genes. The PFGE performed showed that EHEC O157:H7 isolates were genetically diverse, with two minor pulsotypes and eight singletons. The MLST analysis identified three sequence types (STs) (ST10, ST11 and ST1204) that have been previously reported associated with outbreaks. The STs identified in this study pose a potential public health risk to consumers of untreated environmental water and closed human contacts. There is necessity to enhance surveillance in reducing the propagation of this bacterium which is a public health problem.The National Health Laboratory Service (NHLS), National Research Foundation (NRF), Rand Water and University of Pretoria, South Africa.http://www.elsevier.com/locate/scitotenv2020-11-20hj2019Medical Microbiolog

Non-vaccine serotype pneumococcal carriage in healthy infants in South Africa following introduction of the 13-valent pneumococcal conjugate vaccine

BACKGROUND. Pneumococcal carriage studies provide a baseline for measuring the impact of pneumococcal conjugate vaccines (PCVs). The advent of conjugate vaccines has led to reductions in vaccine serotypes (VTs) in pneumococcal carriage. However, increasing non-vaccine serotypes (NVTs) remain a significant concern, necessitating continued surveillance of serotypes in the 13-valent PCV vaccine (PCV13) era. OJECTIVES. To investigate pneumococcal carriage, serotype distribution and risk factors for pneumococcal colonisation among children presenting for routine immunisation at two clinics in Gauteng Province, South Africa (SA), 10 years after PCV introduction into the SA Expanded Programme on Immunisation (EPI-SA). METHODS. Nasopharyngeal swabs were collected from 322 healthy children aged between 6 weeks and 5 years at two clinic centres in 2014 and 2016. Demographic data, risk factors for colonisation and vaccination details were recorded. The pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. RESULTS. Pneumococci were isolated from 138/316 healthy children (43.7%) presenting for routine immunisation at two clinics. The median age was 8.3 months and the age range 1.4 months - 5 years. Carriage varied across the age groups: 6 - 14 weeks 35.5%, 9 months 27.5%, 18 months 21.7%, and 5 years 15.2%. Risk factors significantly associated with pneumococcal colonisation included young age (9 - 18 months (odds ratio OR 3.5; 95% confidence interval (CI) 1.9 - 5.9), type of dwelling (single room (OR 8.1; 95% CI 1.3 - 52.3) or informal dwelling (OR 2.4; 95% CI 1.2 - 4.5)) and Haemophilus influenzae carriage (OR 5.6; 95% CI 0.6 - 2.5). Of the 26 serotypes detected, 19F (10/121; 8.3%) was the most frequent. The most frequent NVTs were 23B (16/121; 13.2%), 15B/C (14/121; 11.6 %) and 35B (11/121; 8.2%). Children aged 9 months carried the highest proportion of NVTs (33/101; 32.7%). Penicillin non-susceptibility was observed in 20 NVT isolates (20/36; 55.6%) and 2 VT isolates (2/36; 5.6%). CONCLUSIONS. The pneumococcal carriage prevalence described in our study varied across the age groups and was lower compared with other African studies that looked at pneumococcal carriage post PCV. The study gave insight into the common NVTs encountered at two immunisation clinics in Gauteng. Given that pneumococcal carriage precedes disease, common colonisers such as 15B/C and 35B may be sufficiently prevalent in carriage for expansion to result in significant disease replacement.NHLS Research Trust Development grant.http://www.samj.org.zadm2022Medical Microbiolog

Genomic analysis of two drug-resistant clinical Morganella morganii strains isolated from UTI patients in Pretoria, South Africa

Morganella morganii is an opportunistic bacterial pathogen of the Enterobacteriaceae family that is occasionally isolated from clinical (animal and human) specimens with varying resistance profiles. Detailed genomic analyses of drug‐resistant M. morganii strains are relatively limited, particularly in Africa, which is also due to their relatively low isolation rates from clinical settings. Here we report on two multidrug‐resistant clinical M. morganii isolates from urine specimens of two hospitalized patients in South Africa who presented with urinary tract infections in 2013. The isolates, M006 and E042, were only susceptible to carbapenems, amikacin and tigecycline. One strain, M006, had a novel class 1 integron, ln1484, associated with aadA7, sul1and gcuD gene cassettes and a Col3M plasmid replicase gene. The ln1484 intI1:aadA7:sul1 genes were bracketed by a TnAs3 composite transposon while a tet(B) gene was found on an IS4 family transposon. The rare blaDHA‐4 and blaDHA‐1 AmpC β‐lactamase genes were identified on the isolates’ chromosome. The isolates were phylogenetically distant and closely related to other international strains, suggesting that they were not obtained from a single epidemiological source. Further molecular surveillance is necessary to establish the prevalence of these MDR strains in the tertiary hospital. Moreover antibiotic stewardship and antibiotic sensitivity testing of all clinical isolates should be undertaken after empirical treatment to inform tailored therapy as well as reduce escalation of resistance and associated morbidities and mortalities.Supplementary Data S1. Metadata of isolates included in phylogenomic analysis.The National Health Laboratory Services (Grant No.: 94445), the University of Pretoria (Grant No.: A0702) and the South African Medical Research Council.https://onlinelibrary.wiley.com/journal/1472765x2021-01-01hj2020Medical Microbiolog

Molecular screening of clinical multidrug-resistant gram-negative bacteria shows endemicity of carbapenemases, coexistence of multiple carbapenemases, and rarity of mcr in South Africa

Please read abstract in the article.The NHLS and the National Research Foundation of South Africa.https://home.liebertpub.com/publications/microbial-drug-resistance/44hj2023Medical Microbiolog