Surveillance of Salmonella spp. in the environment of public hospitals in KwaZulu-Natal, South Africa.

AIM: To investigate the dissemination of Salmonella spp. within four levels of government hospitals in KwaZulu-Natal, South Africa. METHODS: The identification of Salmonella spp. was performed by amplification of the invA gene. Isolates were subjected to antimicrobial susceptibility testing and molecular characterization of eight resistance genes (qnrA, qnrB, qnrS, tetA, tetB, tetC, tetG, ermB) and three virulence genes (sitC, spvA, spv). Genetic relatedness between isolates was determined using enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction. FINDINGS: Ninety-four isolates were obtained. The largest source of isolates was the regional hospital. Paediatric wards had the highest prevalence of isolates. Nurses' tables contained the most isolates out of all sites sampled. Twenty-two clusters indicating diverse isolates were obtained via molecular typing. Four main ERIC types were identified, each unique to a specific hospital. A possibility of dissemination across the wards was noted as highly related isolates were present at various sites within the wards. Many of these sites were highly trafficked areas by healthcare staff. Ten multi-drug-resistant isolates were found. CONCLUSIONS: This study suggests that infection prevention and control strategies that involve environmental cleaning and decontamination may not be enough, or adhered to sufficiently, to prevent the dissemination of Salmonella spp.

In vitro potentiation of carbapenems with tannic acid against carbapenemase-producing enterobacteriaceae: exploring natural products as potential carbapenemase inhibitors.

AIMS: We hypothesized and confirmed that tannic acid (TA) reverses carbapenem resistance by inhibiting carbapenemases in class A and B carbapenemase-producing Enterobacteriaceae. METHODS AND RESULTS: Minimum inhibitory concentrations of carbapenems in the presence and absence of TA and other efflux pump inhibitors, TA-carbapenemases inhibition assays and computational studies showed that TA had the greatest effect on metallo-ß-lactamases (MBLs) followed by class A serine-ß-lactamases (SBLs). TA completely reversed the MICs of MBL producers from between 32 and ≥512 mg l-1 to susceptible values (<4 mg l-1 ) while substantially reducing the MICs of SBLs from between 16 and >512 mg l-1 to <4 to 16 mg l-1 . Tolerable cytotoxic effect was observed for the concentrations tested (8-1024 mg l-1 ). TA inhibited enzymes with a marked difference of ≈50% inhibition (IC50 ) for NDM-1 (270 µmol l-1 ) and KPC-2 (15  µmol l-1 ). CONCLUSION: TA inhibited both MBLs and SBLs by targeting their hydrophobic sites. Moreover, TA had a stronger binding affinity for MBLs than SBLs as the MBLs, specifically VIM-1 (-43·7220 ± 0·4513 kcal mol-1 ) and NDM-1(-44·2329 ± 0·3806 kcal mol-1 ), interact with a larger number of their catalytic active-site residues than that of OXA-48 (-22·5275 ±  0·1300 kcal mol-1 ) and KPC-2 (-22·1164 ± 0·0111 kcal mol-1 ). SIGNIFICANCE AND IMPACT OF THE STUDY: Tannic acid or its analogues could be developed into carbapenemase-inhibiting adjuvants to restore carbapenem activity in CRE infections, save many lives and reduce healthcare associated costs.

Molecular characterization of methicillin-resistant Staphylococcus aureus isolates from a hospital in Ghana

Background: Methicillin-resistant Staphylococcus aureus (MRSA) are a major cause of hospital- and community-acquired infection. They can colonize humans and cause a wide range of infections including pneumonia, endocarditis and bacteraemia. We investigated the molecular mechanism of resistance and virulence of MRSA isolates from a teaching hospital in Ghana. Methodology: A total of 91 S. aureus isolates constituted the initial bacterial sample. Identification of S. aureus was confirmed by the VITEK 2 system. The cefoxitin screen test was used to detect MRSA and antibiotic susceptibility was determined using the VITEK 2 system. The resistance (mecA, blaZ, aac-aph, ermC, and tetK) and virulence (lukS/F-PV, hla, hld and eta) genes were amplified by polymerase chain reaction (PCR) and positive samples subjected to DNA sequencing. Pulsed field gel electrophoresis (PFGE) was used to ascertain the relatedness of the isolates. Results: Fifty-eight of 91 (63.7%) isolates were putatively methicillin resistant by the phenotypic cefoxitin screen test and oxacillin MICs. However, 43 (47%) of the isolates were genotypically confirmed as MRSA based on PCR detection of the mecA gene. Furthermore, 37.9% of isolates displayed resistance to tetracycline, 19% to trimethoprim-sulphamethoxazole, 15.5% to clindamycin, 12.1% to gentamicin, 13.8% to ciprofloxacin and erythromycin, 6.9% to moxifloxacin and 7.0% to rifampicin. None of the isolates was positive for inducible clindamycin resistance. The prevalence of resistance (mecA, blaZ, aac(6')-aph(2''), tetK, and ermC) and virulence (hla and lukS/F-PV) genes respectively were 74%, 33%, 22%, 19%, 3%, 5% and 3%, with isolates organized in two highly related clades. Conclusion: Results indicate a fairly high occurrence of MRSA, which can complicate the effective therapy of S. aureus infections, necessitating surveillance and stringent infection control programmes to forestall its spread

Colistin and tigecycline resistance in carbapenemase-producing Gram-negative bacteria: emerging resistance mechanisms and detection methods.

A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase-producing Gram-negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans-complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT-PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC-FGH-IJK, mexAB-XY-oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr-1 gene conferring resistance to colistin was identified via WGS, trans-complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI-TOF MS, micro-array and real-time multiplex PCR hold much promise for the future as new detection tools.

In vitro evaluation of metal chelators as potential metallo- ß -lactamase inhibitors.

AIMS: This study aimed at investigating the use of metal chelators as potential metallo-ß-lactamase inhibitors (MBL). METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of meropenem was ascertained alone and in combination with various concentrations of macrocyclic (1,4,7- triazacyclononane-1-glutaric acid-4,7-diacetic acid = NODAGA) peptide derivatives and acyclic (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine = TPEN and di-(2-picolyl)amine = DPA) metal chelators using the broth microdilution method. MICs of meropenem against carbapenem-resistant enterobacteriaceae (CRE) producing MBLs were decreased to concentrations as low as 0·06 mg l(-1) in the presence of some metal chelators. TPEN at 4 and 8 mg l(-1) showed the best activity by decreasing meropenem MICs to 0·5 and 0·06 mg l(-1) , respectively, for some New Delhi Metallo-beta-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase (VIM) -producing enterobacteriaceae. DPA at 8 and 16 mg l(-1) was also able to decrease meropenem MICs to 1 and 0·125 mg l(-1) , respectively, for these CREs. NODAGA peptide derivatives showed the least inhibition as 32 mg l(-1) was required for meropenem MICs to be decreased to 0·06 mg l(-1) against an NDM-1 producing isolate. CONCLUSION: The various metal chelators, TPEN, DPA and NODAGA peptide derivatives were able to inhibit the MBLs in decreasing order of activity, rendering CREs susceptible to meropenem. SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of new antibiotics, this study evaluated metal chelators as potential MBL inhibitors.

Prevalence of antibiotic resistance in Campylobacter isolates from commercial poultry suppliers in KwaZulu-Natal, South Africa.

OBJECTIVES: Campylobacter jejuni isolated from broiler and layer chickens from registered abattoirs in KwaZulu-Natal, South Africa, were tested for their susceptibility to eight antibiotics. METHODS: Using agar dilution, susceptibility to eight antibiotics was determined for C. jejuni recovered from the caeca. RESULTS: A total of 155 isolates were collected of which 77 were identified as C. jejuni (broilers n = 56 and layers n = 21). Resistance was highest to tetracycline (broilers 98.2% and layers 100%) and ceftriaxone (broilers 96.4% and layers 100%). High susceptibility was found to ciprofloxacin (broilers 91% and layers 76%) and gentamicin (broilers 98% and layers 81%). Susceptibilities to each of the antibiotics for the broilers and layers, respectively, were: 50% and 57% for erythromycin, 45% and 24% for clarithromycin, 68% and 43% for ampicillin and 64% and 48% for nalidixic acid. Statistically significant differences were detected for the MIC(50) of gentamicin, ciprofloxacin and tetracycline between broilers and layers (P < 0.001) with the MIC(90) of gentamicin also of significant difference (P = 0.01). Multiresistance was detected in 23% and 43% of the isolates from broiler and layer chickens, respectively. CONCLUSIONS: Mass therapy procedures used in animal husbandry have a potential impact on antibiotic resistance development in C. jejuni.